Minimal volume reprogramming of mononuclear cells

ABSTRACT

The invention provides compositions and methods for reprogramming minimal volumes of mononuclear cells. In particular aspects, the invention provides methods and compositions for reprogramming minimal volumes of umbilical cord blood obtained from cord blood segments from cryopreserved cord blood segments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 62/007,924, filed Jun. 4, 2014, which isincorporated by reference in its entirety.

BACKGROUND

1. Technical Field

The invention generally relates to compositions and methods forreprogramming non-pluripotent cells. More particularly, the inventionrelates to reprogramming mononuclear cells from low volume samples ofblood.

2. Description of Related Art

Through their ability to self-renew and differentiate into any somaticcell type, induced pluripotent stem cells (iPSCs) have remarkable, butas yet untapped, therapeutic potential. Blood provides an attractivesource of somatic cells for reprogramming as it is available inrelatively large volumes and is easy to collect. However, blood, likeother sources of explant-derived somatic cells for reprogramming, hassome limitations in therapeutic applications since it must be HLA-typedand screened for pathogens before reprogrammed cells can be administeredto a subject. What is needed in the art therefore is a readilyaccessible source of mononuclear cells for reprogramming that has beenscreened for HLA compatibility, pathogens and genetic abnormalities inthe donor.

SUMMARY OF THE INVENTION

The invention overcomes the limitations in the art by providingcompositions and methods for using cord blood segments from umbilicalcord blood (UCB) units as a source of somatic cells for cellularreprogramming. UCB represents an attractive starting material for thegeneration of allogeneic human induced pluripotent stem cells (hiPSCs)for therapeutic applications since it is an accessible, banked, tested,HLA-typed source of cells with established donor consent and reducedchance of age-related genetic mutations. Because UCB units that havebeen screened in this manner are often cryopreserved for later use by anHLA-compatible recipient, the invention's use of cord blood segmentsprevents the need to thaw UCB units thereby preserving the UCB unit forother applications such as transplantation therapy or furtherreprogramming.

Cord blood segments are segments of tubing that form an integral part ofa cord blood bag and contain a portion of UCB from the cord blood unit.Cord blood segments are formed when the tubing is sealed in sections toprovide small volumes or “segments” of UCB. Currently, cord bloodsegments of cryopreserved UCB are only used for confirmatory anddiagnostic testing purposes. The minimal volume in these frozen segmentsrepresents a major challenge for culturing and further processing sincethe number of mono-nucleated cells present in such samples is relativelysmall. This difficulty is compounded in cellular reprogramming contextssince cellular reprogramming itself is an inefficient process,especially when attempting non-integrative methods and without the useof oncogenes such as KLF4 and MYC.

The invention overcomes these difficulties by providing methods andcompositions for reprogramming mononuclear cells from minimal volumes ofscreened blood to derive human induced pluripotent stem cells that aresuitable for transplantation.

It is therefore an object of the invention to provide a method ofproducing induced pluripotent stem cells (iPSC) from blood comprisingproviding a minimal volume of blood comprising mononuclear cells, andreprogramming the mononuclear cells to iPSC by introducing to themononuclear cells at least one polynucleotide encoding least onereprogramming factor polypeptide or introducing to the mononuclear cellsat least one reprogramming factor polypeptide.

A further object of the invention is to provide a method of producingiPSC from umbilical cord blood comprising: providing a sample ofcryopreserved umbilical cord blood comprising mononuclear cells, whereinthe sample is between about 1 mL and about 10 μL in volume; introducingto the mononuclear cells a composition comprising (i) at least onepolynucleotide encoding at least one reprogramming factor polypeptide,or (ii) at least one reprogramming factor polypeptide; contacting themononuclear cells with at least one of a GSK3 inhibitor, a MEKinhibitor, a ROCK inhibitor, and a TGFβR inhibitor; and culturing thecontacted mononuclear cells in a culture medium comprising a GSK3inhibitor, a MEK inhibitor, and a ROCK inhibitor, but not a TGFβRinhibitor.

In some aspects of the invention, the cryopreserved umbilical cord bloodis obtained from a segment from an umbilical cord unit.

A further object of the invention is to provide a composition comprising(i) between about 1 mL and about 10 μL of umbilical cord blood orperipheral blood, and (ii) at least one exogenous polynucleotideencoding at least one reprogramming factor polypeptide, or at least oneexogenous reprogramming factor polypeptide.

In some aspects of the invention, the composition comprises at least oneof a GSK3 inhibitor, a MEK inhibitor, a ROCK inhibitor, and a TGFβRinhibitor.

A further objective of the invention is to provide a compositioncomprising between about 100 and 1×10⁴ mononuclear cells, and (i) atleast one exogenous polynucleotide encoding at least one reprogrammingfactor polypeptide, or (ii) at least one exogenous reprogramming factorpolypeptide.

In some aspects of the invention, the composition comprises less thanabout 1×10⁴ mononuclear cells.

In some aspects of the invention, the composition comprises at least oneof a GSK3 inhibitor, a MEK inhibitor, a ROCK inhibitor, and a TGFβRinhibitor.

A further object of the invention is to provide a kit for producing iPSCwherein the kit comprises a minimal volume of blood, and (i) at leastone polynucleotide encoding at least one reprogramming factorpolypeptide, or (ii) at least one reprogramming factor polypeptide.

In some aspects of the invention, the blood comprises a segment from anumbilical cord unit.

In some aspects of the invention, the kit comprises at least one of aGSK3 inhibitor, a MEK inhibitor, a ROCK inhibitor, and a TGFβRinhibitor.

A further object of the invention is to provide a kit for producing iPSCwherein the kit comprises between about 100 and 1×10⁴ mononuclear cells,and (i) at least one polynucleotide encoding at least one reprogrammingfactor polypeptide, or (ii) at least one reprogramming factorpolypeptide.

In some aspects of the invention, the kit comprises less than about1×10⁴ mononuclear cells.

In some aspects of the invention, the kit comprises at least one of aGSK3 inhibitor, a MEK inhibitor, a ROCK inhibitor, and a TGFβRinhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of an embodiment for producing humaninduced pluripotent stem cells (hiPSCs) from UCB segments andsubsequent, characterization and banking. A segment of banked umbilicalcord blood, such as 100 μL in volume, is Ficoll treated for mononuclearcells and expanded for 1-2 weeks in hematopoietic stem cell expansionmedium. The culture is further enriched for stem and progenitorpopulation and induced to reprogram using, for example, anon-integrative strategy to express OCT4 and SOX2 in defined culture.After 2 weeks in culture, reprogrammed cells expressingSSEA4/TRA181/CD30 are directly sorted into 96-well plates. IndividualhiPSC colonies are then expanded in defined culture and selected clonesare optionally banked as HLA-matched allogeneic lines for potentialtherapeutic use.

FIGS. 2A-2E: CD34 enrichment and culture post segment harvest. FIG. 2A)CD34 positive cells derived from segments by FACS transferred to culturefor expansion. FIG. 2B) Sorted cells efficiently expand in cultureconditions. FIG. 2C) Appearance and FIG. 2D) flow profile of culturedcells. A majority of the cells retained their stem cell property asdemonstrated by large population subset expressing CD34. FIG. 2E)Cultured cells are readily transfected as demonstrated with GFP.

FIGS. 3A and 3B: Reprogramming of starting blood cells. Flow cytometryprofile of reprogramming pools of FIG. 3A) cord blood segment of FIG.3B) peripheral blood after 2 weeks post induction with a non-integratingmethod expressing OCT4 and SOX2.

FIGS. 4A-4E. Episomal reprogrammed hiPSCs are readily sorted asindividual cells, maintain their undifferentiated state, and are free oftransgenes. FIG. 4A) Flow cytometry sorting of episomal inducedreprogramming of various starting cell lines reprogrammed in feedercell-free culture directly added to 96-well plate. FIG. 4B) qRT-PCR forNANOG expression for each well of a SSEA4/TRA181/CD30 direct sorted(FACS) 96-well plate. FIG. 4C) Pluripotency markers detected byimmunofluorescence. FIG. 4D) PCR analysis for episomal DNA derived fromvarious hiPSC clones. Lanes 1-6, derived clonal hiPSC lines; Lane 7, aline maintaining episomal constructs used a positive control; Lane 8,untransfected starting line; Lane 9, hiPSC generated using lentiviralconstructs (to serve as a control against cross contamination); Lane 10,episomal vector used as positive control. Input of 100 ng genomic DNAand 30 PCR cycles were used for all sets. FIG. 4E) Flow cytometryprofile for selected hiPSC clones from various parental lines. Upper rowprofiles SSEA4/TRA181 surface expression. Bottom row profiles OCT4/NANOGintracellular expression.

FIGS. 5A-5D: Genomic stability and pluripotency are maintained duringcontinuous single cell and feeder-free culture. FIG. 5A) Flow cytometryprofile and cytogenetic analysis of long-term passaged hiPSC clones infeeder-free and single cell enzymatically passaged culture. FIG. 5B)Copy number variation as assessed by array comparative genomichybridization and single nucleotide polymorphism. FIG. 5C) Embryoid bodyformation and differentiation. Immunocytochemistry conducted 28 dayspost differentiation: Ectoderm, Tujl; Mesoderm, alpha smooth muscleactin (aSMA); Endoderm, AFP. FIG. 5D) Histological sections of teratomaderived from hiPSCs. Black arrows, endoderm; white arrows, ectoderm;gray arrows, mesoderm.

FIGS. 6A-6C. Generated hiPSCs resemble cells associated with the groundstate of pluripotency. FIG. 6A) Heatmap derived from a Fluidigm dynamicarray depicting relative gene expression levels of pluripotency anddifferentiation genes of conventionally grown cells versus cells grownin FMM. FIG. 6B) Hierarchical clustering on the 339 probe sets using acomplete linkage method based on Euclidean distance measurements. FIG.6C) Representative images of HEK27me3 in a hiPSC clone maintained in FMMmedia or adapted to conventional culture for 5 passages.

FIGS. 7A-7F shows cloning maps illustrating examples of the lentiviralconstructs (FIG. 7A-7B) and episomal constructs (FIG. 7C-7F) used forreprogramming. Lentiviral constructs include an EF1α promoter and a LOXPsite for CRE-mediated excision of transgenes. Episomal constructs alsoinclude an EF1α promoter.

DEFINITIONS

The articles “a,” “an,” and “the” are used herein to refer to one or tomore than one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The use of the alternative (e.g., “or”) should be understood to meaneither one, both, or any combination thereof of the alternatives.

The term “and/or” should be understood to mean either one, or both ofthe alternatives.

As used herein, the term “substantially” or “essentially” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that is about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% or higher compared to a reference quantity, level,value, number, frequency, percentage, dimension, size, amount, weight orlength. In one embodiment, the terms “essentially the same” or“substantially the same” refer a range of quantity, level, value,number, frequency, percentage, dimension, size, amount, weight or lengththat is about the same as a reference quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length.

As used herein, the terms “substantially free of” and “essentially freeof” are used interchangeably, and when used to describe a composition,such as a cell population or culture media, refer to a composition thatis free of a specified substance, such as, 95% free, 96% free, 97% free,98% free, 99% free of the specified substance, or is undetectable asmeasured by conventional means. Similar meaning can be applied to theterm “absence of,” where referring to the absence of a particularsubstance or component of a composition.

As used herein, the term “appreciable” refers to a range of quantity,level, value, number, frequency, percentage, dimension, size, amount,weight or length or an event that is readily detectable by one or morestandard methods. The terms “not-appreciable” and “not appreciable” andequivalents refer to a range of quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length or anevent that is not readily detectable or undetectable by standardmethods. In one embodiment, an event is not appreciable if it occursless than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001% or less of the time.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. In particular embodiments, the terms “include,”“has,” “contains,” and “comprise” are used synonymously.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of.” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present.

By “consisting essentially of” is meant including any elements listedafter the phrase, and limited to other elements that do not interferewith or contribute to the activity or action specified in the disclosurefor the listed elements. Thus, the phrase “consisting essentially of”indicates that the listed elements are required or mandatory, but thatno other elements are optional and may or may not be present dependingupon whether or not they affect the activity or action of the listedelements.

Reference throughout this specification to “one embodiment,” “anembodiment,” “a particular embodiment,” “a related embodiment,” “acertain embodiment,” “an additional embodiment,” or “a furtherembodiment” or combinations thereof means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,the appearances of the foregoing phrases in various places throughoutthis specification are not necessarily all referring to the sameembodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

As used herein, the term “about” or “approximately” refers to aquantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length that varies by as much as 30, 25, 20, 25, 10,9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value,number, frequency, percentage, dimension, size, amount, weight orlength. In particular embodiments, the terms “about” or “approximately”when preceding a numerical value indicates the value plus or minus arange of 15%, 10%, 5%, or 1%, or any intervening range thereof.

As used herein, the term “mononuclear cell” refers to a cell found inblood that has a single, round nucleus.

As used herein, the term “pluripotent” refers to the ability of a cellto form all lineages of the body or soma (i.e., the embryo proper). Forexample, an embryonic stem cell is a type of pluripotent stem cell thatis able to form cells from each of the three germs layers: the ectoderm,the mesoderm, and the endoderm. Pluripotency can be determined, in part,by assessing pluripotency characteristics of the cells. Pluripotencycharacteristics include, but are not limited to: (i) pluripotent stemcell morphology; (ii) the potential for unlimited self renewal (iii)expression of pluripotent stem cell markers including, but not limitedto SSEA1 (mouse only), SSEA3/4; SSEA5, TRA1-60/81; TRA1-85, TRA2-54,GCTM-2, TG343, TG30, CD9, CD29, CD133/prominin, CD140a, CD56, CD73,CD90, CD105, OCT4, NANOG, SOX2, CD30 and/or LD50; (iv) ability todifferentiate to all three somatic lineages (ectoderm, mesoderm andendoderm) (v) teratoma formation consisting of the three somaticlineages; and (vi) formation of embryoid bodies consisting of cells fromthe three somatic lineages.

As used herein, the term “non-pluripotent cell” refers to any cell thatdoes not possess full pluripotency, such as incompletely or partiallypluripotent stem cells, multipotent cells, oligopotent cells, unipotentcells (e.g. progenitor cells), and terminally differentiated cells.

As used herein, the terms “reprogramming” or “dedifferentiation” or“increasing cell potency” or “increasing developmental potency” refersto a method of increasing the potency of a cell or dedifferentiating thecell to a less differentiated state. For example, a cell that has anincreased cell potency has more developmental plasticity (i.e., candifferentiate into more cell types) compared to the same cell in thenon-reprogrammed state. In other words, a reprogrammed cell is one thatis in a less differentiated state than the same cell in anon-reprogrammed state.

As used herein, the term “introducing” refers to a process thatcomprises contacting a cell with a polynucleotide, polypeptide, or smallmolecule. An introducing step may also comprise microinjection ofpolynucleotides or polypeptides into the cell, use of liposomes todeliver polynucleotides or polypeptides into the cell, or fusion ofpolynucleotides or polypeptides to cell permeable moieties to introducethem into the cell.

As used herein, the term “reprogramming efficiency” refers to the numberof cells in a sample that are successfully reprogrammed to pluripotentcyrelative to the total number of cells in the sample. Reprogrammingefficiency may be measured as a function of pluripotency markers. Suchpluripotency markers include, but are not limited to, the expression ofpluripotency marker proteins and mRNA, pluripotent cell morphology andcolony formation. For example, a reprogramming efficiency of 5%indicates that 5% of the cells in a sample of cells co-expresses SSEA4and TRA-181/160.

As used herein, “culture” or “cell culture” refers to the maintenance,growth and/or differentiation of cells in an in vitro environment. “Cellculture media,” “culture media” (singular “medium” in each case),“supplement” and “media supplement” refer to nutritive compositions thatcultivate cell cultures.

As used herein, “cultivate” refers to the sustaining, propagating(growing) and/or differentiating of cells outside of tissue or the body,for example in a sterile plastic (or coated plastic) cell culture dishor flask. “Cultivation” may utilize a culture medium as a source ofnutrients, hormones and/or other factors helpful to propagate and/orsustain the cells.

As used herein, the terms “enrich,” “enriching,” “select” and“selecting” are used interchangeably herein to refer to increasing theamount of a specified component in a composition, such as a compositionof cells, and “enriched”, when used to describe a composition of cellssuch as a cell population, refers to a population of cells having anincreased amount proportionally of a specified component as compared tothe proportion of such component in the population of cells prior tobeing enriched. For example, a composition such as a population of cellsmay be enriched with respect to a target cell type (i.e., cells havingspecified characteristics), thus having an increased proportion orpercent of the target cell type as compared to the proportion of thetarget cells present in the population of cells before being enriched. Apopulation of cells may be enriched for a target cell type by cellselection and sorting methods known in the art. In some embodiments, apopulation of cells is enriched by a sorting or selection process asdescribed in the examples herein. In a particular embodiment, a methodthat enriches for a target cell population enriches the cell populationwith respect to the target cell population by at least about 20%,meaning that the enriched cell population comprises proportionatelyabout 20% more of the target cell type than in the population before thepopulation was enriched. In one embodiment, a method that enriches for atarget cell population enriches the cell population with respect to thetarget cell population proportionately by at least about 30+%, 40+%,50+%, 60+%, 70+%, 80%, 85%, 90%, 95%, 97%, 98% or 99%, or at least about98%, or in particular embodiments, about 99%.

“Isolate” or “isolating” refers to separating and collecting acomposition or material from its natural environment, such as theseparating of individual cell or cell cultures from tissue or the body.In one aspect, a population or composition of cells is substantiallyfree of cells and materials with which it can be associated in nature.“Isolated” or “purified” or “substantially pure”, with respect to atarget population of cells, refers to a population of cells that is atleast about 50%, at least about 75%, at least about 85%, at least about90%, and in particular embodiments, at least about 95% pure, withrespect to the target cells making up a total cell population. Purity ofa population or composition of cells can be assessed by appropriatemethods that are well known in the art. For example, a substantiallypure population of pluripotent cells refers to a population of cellsthat is at least about 50%, at least about 75%, at least about 85%, atleast about 90%, and in particular embodiments at least about 95%, andin certain embodiments about 98% pure, with respect to pluripotent cellsmaking up the total cell population. The term “essentially pure” is usedinterchangeably herein with “substantially pure”.

Two types of pluripotency have previously been described: the “primed”or “metastable” state of pluripotency akin to the epiblast stem cells(EpiSC) of the late blastocyst and the “Naïve” or “Ground” state ofpluripotency akin to the inner cell mass of the early/preimplantationblastocyst. While both pluripotent states exhibit the characteristics asdescribed above, the naïve or ground state further exhibits; (i)preinactivation or reactivation of the X-chromosome in female cells (ii)improved clonality and survival during single-cell culturing (iii)global reduction in DNA methylation, (iv) reduction of H3K27me3repressive chromatin mark deposition on developmental regulatory genepromoters, and (v) reduced expression of differentiation markersrelative to primed state pluripotent cells. Standard methodologies ofcellular reprogramming in which exogenous pluripotency genes areintroduced to a somatic cell, expressed and then either silenced orremoved from the resulting pluripotent cells are generally seen to havecharacteristics of the primed-state of pluripotency. Under standardpluripotent cell culture conditions such cells remain in the primedstate unless the exogenous transgene expression is maintained, whereincharacteristics of the ground-state are observed.

As used herein, the phrase “human leukocyte antigens matched,” or“HLA-matched,” refers to the immunological compatibility of the humanleukocyte antigens between a donor of a tissue or cell sample, and therecipient of the tissue or cell sample.

As used herein, the term “cord blood segment,” or “segment,” refers to acompartment on a cord blood freezing bag that is typically used forproducing detachable aliquots of cord blood for confirmatory testing.The terms “cord blood segment” and “segment” may also refer to the blood(or other material) contained within such compartment.

“Primary culture” refers to cells, tissue and/or culture where theisolated cells are placed in a first culture vessel with culture medium.The cells, tissue and/or culture may be sustained and/or mayproliferate, however, as long as the cells, tissue and/or culture remainin the first vessel the cells, tissue and/or culture are referred to asthe primary culture.

As used herein, the term “non-integrative reprogramming” refers to amethod of reprogramming a non-pluripotent stem cell by introducing oneor more reprogramming factors by any means that does not result in anexogenous polynucleotide becoming part of or integrated into thereprogrammed cell genome. Non-integrative reprogramming may also bereferred to as “footprint-free reprogramming.”

As used herein, the term “minimal volume” refers to a volume that isabout, or less than, 1 ml. Some non-limiting embodiments of a minimalvolume include, but are not limited to, about, or less than, 900 μl, 800μl, 700 μl, 600 μl, 500 μl, 400 μl, 300 μl, 200 μl, 100 μl or 50 μl, aswell as about, or less than, any volume intervening these volumes.

As used herein, the term “cryopreservation” refers to a process ofcooling and storing cells, tissues, or organs at low temperatures tomaintain their viability. For example, cryopreservation includes thecooling and storing of cells, tissues, or organs at or below freezing(i.e. 0° C.).

As used herein, the term “cryopreserved” refers to a material that hasbeen subjected to cryopreservation.

As used herein, the term “single cell culture” refers to the culture andexpansion of cells from non-aggregated, individual cells.

As used herein, the term “reprogrammed” refers to a somatic cell thathas been reprogrammed to an induced pluripotent stem cell (iPSC).

As used herein, a “feeder-free” (FF) environment refers to anenvironment such as a cell culture or culture media essentially free offeeder cells and/or which has not been pre-conditioned by thecultivation of feeder cells. “Pre-conditioned” medium refers to a mediumharvested after feeder cells have been cultivated within the medium fora period of time, such as for at least one day. Pre-conditioned mediumcontains many mediator substances, including growth factors andcytokines secreted by the feeder cells cultivated in the medium.

The terms “small molecule reprogramming agent” or “small moleculereprogramming compound” are used interchangeably herein and refer tosmall molecules that can increase developmental potency of a cell,either alone or in combination with other pluripotency factors. A “smallmolecule” refers to an agent that has a molecular weight of less thanabout 5 kD, less than about 4 kD, less than about 3 kD, less than about2 kD, less than about 1 kD, or less than about 0.5 kD. Small moleculesinclude, but are not limited to: nucleic acids, peptidomimetics,peptoids, carbohydrates, lipids or other organic or inorganic molecules.Libraries of chemical and/or biological mixtures, such as fungal,bacterial, or algal extracts, are known in the art and can be used as asource of small molecules in certain embodiments. In particularembodiments, the small molecule reprogramming agent used herein has amolecular weight of less than 10,000 daltons, for example, less than8,000, 6,000, 4,000, 2,000 daltons, e.g., between 50-1,500, 500-1,500,200-2,000, 500-5,000 daltons.

DETAILED DESCRIPTION

The present invention relates to methods and compositions forreprogramming mononuclear cells to induced pluripotent stem cells (iPSC)using minimal volumes of blood. Mononuclear cells capable of beingreprogrammed form a small portion of the cells found in the blood. Thus,minimal volumes of blood provide few cells capable of beingreprogrammed. The difficulty of reprogramming cells from minimal volumesof blood is further complicated by the relative inefficiency of knownreprogramming methods which typically fail to reprogram a large numberof cells in a given sample. Despite these challenges, the inventorssurprisingly discovered that efficient reprogramming of mononuclearcells from minimal volumes of blood can be accomplished through the useof the methods and compositions herein.

The development and use of UCB cryostorage technology and cord bloodsegments represents a unique opportunity to reprogram cells of apredefined identity. By reprogramming minimal volumes of UCB found incord blood segments, the invention provides prequalified cGMP-gradeHuman Leukocyte Antigens (HLA)-matched hiPSCs that can be banked andapplied to any number of downstream differentiated allogeneic celltherapies. Such allogeneic hiPSC banks could be created with appropriateHLA diversity to enable application to the broadest patient population.Accordingly, UCB represents an attractive starting material source forthe generation of allogeneic hiPSCs for therapeutic applications: theyare an accessible, banked, tested, HLA-typed source of cells withestablished donor consent and reduced chance of age-related geneticmutations. The development and use of UCB cryostorage technology thatincludes sampling segments represents a unique opportunity to reprogramcells of a predefined identity without compromising the main UCB unititself. UCB unit sampling segments typically consist of 100 μL volumewith minimal number of mononuclear cells. In some aspects, the inventionprovides a reprogramming platform comprising a multistage process forUCB segment CD34⁺ cell selection, expansion, non-integrating cellularreprogramming and scalable iPSC culture. The platform can comprisestage-specific media formulations containing unique small moleculecocktails for enhancing the expansion and reprogramming of mononuclearcells and the maintenance and banking of reprogrammed mononuclear cells(e.g. iPSC). iPSCs reprogrammed according to the methods andcompositions of the invention can be free of transgene, readily culturedand expandable as single cells while maintaining a homogenous andgenomically stable pluripotent population. iPSCs generated or maintainedin the media compositions described herein can exhibit propertiesassociated with the ground state of pluripotency including enhanced cellviability and stability in single cell applications.

Mononuclear cells for use with the invention may be obtained from anysource that provides cells capable of being reprogrammed using themethods and compositions disclosed herein. Mononuclear cells may beobtained from blood, a primary culture of mononuclear cells, or a clonalcell line, for example. Suitable sources of blood include, but are notlimited to, peripheral blood, placental blood and umbilical cord blood(UCB). Peripheral blood may be mobilized according to methods known inthe art. Blood for use with the invention may be fresh or cryopreservedand may be human or non-human in origin. Non-human sources of blood,include, but are not limited to, non-human primate, mouse, rat, horse,pig, bovine and avian sources. In a specific, non-limiting embodiment ofthe invention, mononuclear cells from human cord blood are reprogrammedto iPSC. In another specific, non-limiting embodiment of the invention,mononuclear cells from a segment of cryopreserved human cord blood thatis suitable for transplantation (e.g. screened for pathogens and HLAtyped for matching with an intended recipient) is used for reprogrammingmononuclear cells to iPSC. Thus, mononuclear cells reprogrammed from UCBcan be used in cell therapy.

In some aspects of the invention, mononuclear cells for reprogrammingare obtained from the segment of a UCB cryopreservation bag containingUCB, such as a cord blood unit intended for transplantation. UCB bloodmay be fresh or cryroperserved. In embodiments where the segment of UCBis obtained from a cryopreserved cord blood unit, the invention canprovide a sufficient number of mononuclear cells for reprogramming,while maintaining the remainder of the cord blood unit in acryopreserved state. Thus, the invention allows the mononuclear cellsfrom a cryopreserved cord blood unit to be evaluated for theirreprogramming and therapeutic potential, while maintaining the cordblood unit as a source of mononuclear cells for further reprogramming,testing, research and/or therapeutic applications.

Mononuclear cells may be derived from any source that can provide cellscapable of being reprogrammed according to the methods and compositionsdisclosed herein, and may be heterogeneous or homogeneous with respectto cell types or state of pluripotency. Mononuclear cells may be a“mixed” population of cells comprising cells of varying degrees ofdevelopmental potency. Suitable mononuclear cells for reprogrammingaccording to the invention include, but are not limited to,non-pluripotent cells, incompletely or partially pluripotent stem cells,multipotent cells, oligopotent cells, unipotent cells, terminallydifferentiated cells, or a mixed population of cells comprising anycombination of the foregoing. Some non-limiting examples of mononuclearcells that may be reprogrammed as disclosed herein include, but are notlimited to, hematopoetic stem and progenitor cells (e.g. CD34+ cells),CD45+/CD34+/Lineage− cells, CD45+/Lineage− cells, myeloid progenitorcells, lymphoid progenitor cells, mast cells, monocytes, NK cells,lymphocytes, cytotoxic T cells, helper T cells, regulatory T cells,natural killer T cells, memory T cells, B-cells (e.g. plasma B cells,memory B cells, B-1 cells and B-2 cells), neutrophils, macrophages,basophils, dendritic cells, eosinophils or a combination thereof.

One aspect of the invention relates to compositions for reprogrammingmononuclear cells to iPSC. In some embodiments, mononuclear cells arereprogrammed by introducing to the mononuclear cells polynucleotidesthat encode one or more of the polypeptides OCT4, SOX2, NANOG, KLF4,LIN28, c-MYC, SV40LT, hTERT, SALL4, GLIS, ESRRB, DPPA2, ECAT1, SOX1,SOX3, KLF2, KLF5, 1-MYC, n-MYC, LRH1, UTF1, TBX3, TFCP2L1, SOCS3, STAT3,and TEX10. In one non-limiting embodiment of the invention, mononuclearcells are reprogrammed with one or more polynucleotides encoding OCT4,SOX2, SV40LT, but not KLF4 or c-Myc. In another non-limiting embodiment,mononuclear cells are reprogrammed by introducing polynucleotidesencoding OCT4, SOX2 and SV40LT, but not c-MYC or KLF4. In anothernon-limiting embodiment, mononuclear cells are reprogrammed byintroducing OCT4, SOX2 and SV40LT, but not c-MYC or KLF4. In anothernon-limiting embodiment, mononuclear cells are reprogrammed byintroducing polynucleotides encoding TBX3, TFCP2L1, and SOCS3, but notc-MYC or KLF4. In another non-limiting embodiment, mononuclear cells arereprogrammed by introducing polynucleotides encoding OCT4, SOX2, c-MYCand KLF4. Reprogramming may be accomplished by introducing one or morecopies of the reprogramming factors disclosed herein. The inventionfurther contemplates introducing the above reprogramming factors tocells in the form of a polypeptide and/or mRNA.

In particular embodiments, one or more polynucleotides encoding 1, 2, 3,4, 5 or more copies of one or more of the reprogramming factors selectedfrom the group consisting of: OCT4, SOX2, NANOG, KLF4, LIN28, c-MYC,SV40LT, hTERT, SALL4, GLIS, ESRRB, DPPA2, ECAT1, SOX1, SOX3, KLF2, KLF5,1-MYC, n-MYC, LRH1, UTF1, TBX3, TFCP2L1, SOCS3, STAT3, and TEX10 may beintroduced into a mononuclear cell to reprogram the cell. The copynumber of each reprogramming factor introduced into the cell may be thesame or different in any combination suitable to achieve reprogramming.

In particular embodiments, one or more polynucleotides may be arrangedin any suitable order within a larger polynucleotide, such as a vector(e.g. polycistronic vector). In some embodiments, the vector is anepisomal vector.

The polynucleotides contemplated herein, regardless of the length of thecoding sequence itself, may be combined with other DNA sequences, suchas expression control sequences, promoters and/or enhancers,untranslated regions (UTRs), Kozak sequences, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, internalribosomal entry sites (IRES), recombinase recognition sites (e.g., LoxP,FRT, and Att sites), termination codons, transcriptional terminationsignals, and polynucleotides encoding self-cleaving polypeptides,epitope tags, as disclosed elsewhere herein or as known in the art, suchthat their overall length may vary considerably. It is thereforecontemplated that a polynucleotide fragment of almost any length may beemployed, with the total length preferably being limited by the ease ofpreparation and use in the intended recombinant DNA protocol.

Polynucleotides can be prepared, manipulated and/or expressed using anyof a variety of well established techniques known and available in theart. In order to express a desired polypeptide, a nucleotide sequenceencoding the polypeptide, can be inserted into appropriate vector.Examples of vectors are plasmid, autonomously replicating sequences, andtransposable elements. Additional exemplary vectors include, withoutlimitation, plasmids, phagemids, cosmids, artificial chromosomes such asyeast artificial chromosome (YAC), bacterial artificial chromosome(BAC), or P1-derived artificial chromosome (PAC), bacteriophages such aslambda phage or M13 phage, and animal viruses. Examples of categories ofanimal viruses useful as vectors include, without limitation, retrovirus(including lentivirus), adenovirus, Sendai virus, adeno-associatedvirus, herpesvirus (e.g., herpes simplex virus), poxvirus, baculovirus,papillomavirus, and papovavirus (e.g., SV40). Examples of expressionvectors are pClneo vectors (Promega) for expression in mammalian cells;pLenti4/V5-DEST™, pLenti6N5-DEST™, and pLenti6.2/V5-GW/lacZ (Invitrogen)for lentivirus-mediated gene transfer and expression in mammalian cells.In particular embodiments, coding sequences of polypeptides disclosedherein can be ligated into such expression vectors for the expression ofthe polypeptides in mammalian cells.

In particular embodiments, the vector is an episomal vector or a vectorthat is maintained extrachromosomally. As used herein, the term“episomal” refers to a vector that is able to replicate withoutintegration into host's chromosomal DNA and without gradual loss from adividing host cell also meaning that said vector replicatesextrachromosomally or episomally. The vector is engineered to harbor thesequence coding for the origin of DNA replication or “ori” from alymphotrophic herpes virus or a gamma herpesvirus, an adenovirus, SV40,a bovine papilloma virus, or a yeast, specifically a replication originof a lymphotrophic herpes virus or a gamma herpesvirus corresponding tooriP of EBV. In a particular aspect, the lymphotrophic herpes virus maybe Epstein Barr virus (EBV), Kaposi's sarcoma herpes virus (KSHV),Herpes virus saimiri (HS), or Marek's disease virus (MDV). Epstein Barrvirus (EBV) and Kaposi's sarcoma herpes virus (KSHV) are also examplesof a gamma herpesvirus. Typically, the host cell comprises the viralreplication transactivator protein that activates the replication.

“Expression control sequences,” “control elements,” or “regulatorysequences” present in an expression vector are those non-translatedregions of the vector—origin of replication, selection cassettes,promoters, enhancers, translation initiation signals (Shine Dalgarnosequence or Kozak sequence) introns, a polyadenylation sequence, 5′ and3′ untranslated regions—which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including ubiquitous promoters and inducible promoters may be used.

Illustrative ubiquitous expression control sequences suitable for use inparticular embodiments of the invention include, but are not limited to,a cytomegalovirus (CMV) immediate early promoter, a viral simian virus40 (SV40) (e.g., early or late), a Moloney murine leukemia virus (MoMLV)LTR promoter, a Rous sarcoma virus (RSV) LTR, a herpes simplex virus(HSV) (thymidine kinase) promoter, H5, P7.5, and P11 promoters fromvaccinia virus, an elongation factor 1-alpha (EF1a) promoter, earlygrowth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL),Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translationinitiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5),heat shock protein 90 kDa beta, member 1 (HSP90B1), heat shock protein70 kDa (HSP70), β-kinesin (β-KIN), the human ROSA 26 locus (Irions etal., Nature Biotechnology 25, 1477-1482 (2007)), a Ubiquitin C promoter(UBC), a phosphoglycerate kinase-1 (PGK) promoter, a cytomegalovirusenhancer/chicken β-actin (CAG) promoter, and a β-actin promoter.

Illustrative examples of inducible promoters/systems include, but arenot limited to, steroid-inducible promoters such as promoters for genesencoding glucocorticoid or estrogen receptors (inducible by treatmentwith the corresponding hormone), metallothionine promoter (inducible bytreatment with various heavy metals), MX-1 promoter (inducible byinterferon), the “GeneSwitch” mifepristone-regulatable system (Sirin etal., 2003, Gene, 323:67), the cumate inducible gene switch (WO2002/088346), tetracycline-dependent regulatory systems, etc.

Conditional expression can also be achieved by using a site specific DNArecombinase. According to certain embodiments of the invention,polynucleotides comprise at least one (typically two) site(s) forrecombination mediated by a site specific recombinase. As used herein,the terms “recombinase” or “site specific recombinase” include excisiveor integrative proteins, enzymes, co-factors or associated proteins thatare involved in recombination reactions involving one or morerecombination sites (e.g., two, three, four, five, six, seven, eight,nine, ten or more), which may be wild-type proteins (see Landy, CurrentOpinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives(e.g., fusion proteins containing the recombination protein sequences orfragments thereof), fragments, and variants thereof. Illustrativeexamples of recombinases suitable for use in particular embodiments ofthe present invention include, but are not limited to: Cre, Int, IHF,Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD,TnpX, Hjc, Gin, SpCCE1, and ParA.

In particular embodiments, polynucleotides contemplated herein, includeone or more polynucleotides that encode one or more polypeptides. Inparticular embodiments, to achieve efficient translation of each of theplurality of polypeptides, the polynucleotide sequences can be separatedby one or more IRES sequences or polynucleotide sequences encodingself-cleaving polypeptides. As used herein, an “internal ribosome entrysite” or “IRES” refers to an element that promotes direct internalribosome entry to the initiation codon, such as ATG, of a cistron (aprotein encoding region), thereby leading to the cap-independenttranslation of the gene. See, e.g., Jackson et al., 1990. Trends BiochemSci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000.Examples of IRES generally employed by those of skill in the art includethose described in U.S. Pat. No. 6,692,736. Further examples of “IRES”known in the art include, but are not limited to IRES obtainable frompicornavirus (Jackson et al., 1990).

The polynucleotides contemplated herein may be engineered to provide aself-cleaving peptide through the incorporate of a protease cleavagesite. Suitable protease cleavages sites and self-cleaving peptides areknown to the skilled person (see, e.g., in Ryan et al., 1997. J. Gener.Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594).Exemplary protease cleavage sites include, but are not limited to thecleavage sites of potyvirus NIa proteases (e.g., tobacco etch virusprotease), potyvirus HC proteases, potyvirus P1 (P35) proteases,byovirus NIa proteases, byovirus RNA-2-encoded proteases, aphthovirus Lproteases, enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3Cproteases, comovirus 24K proteases, nepovirus 24K proteases, RTSV (ricetungro spherical virus) 3C-like protease, PYVF (parsnip yellow fleckvirus) 3C-like protease, heparin, thrombin, factor Xa and enterokinase.

In certain embodiments, the self-cleaving polypeptide site comprises a2A or 2A-like site, sequence or domain (Donnelly et al., 2001. J. Gen.Virol. 82:1027-1041). In a particular embodiment, the viral 2A peptideis an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus2A peptide.

In one embodiment, the viral 2A peptide is selected from the groupconsisting of: a foot-and-mouth disease virus (FMDV) 2A peptide, anequine rhinitis A virus (ERAV) 2A peptide, a Thosea asigna virus (TaV)2A peptide, a porcine teschovirus-1 (PTV-1) 2A peptide, a Theilovirus 2Apeptide, and an encephalomyocarditis virus 2A peptide.

One aspect of the invention relates to preparing a minimal volume ofblood for reprogramming the mononuclear cells contained therein. In onenon-limiting embodiment of the invention, reprogramming factors areintroduced directly to the minimal volume of blood. The reprogrammedmononuclear cells (e.g. iPSC) thus obtained may then be seriallyexpanded or selected according to methods known in the art, such asFACS. In another non-limiting embodiment of the invention, the minimalvolume of blood is subjected to methods for purifying the mononuclearcells, such as the removal of erythrocytes by a Ficoll gradient. Thepurified mononuclear cells may then be (i) reprogrammed and thenexpanded and optionally subjected to selection methods (e.g. FACS) toobtain a either homogenous population of iPSC or fully clonal iPSCpopulation, (ii) expanded and subsequently reprogrammed and thenoptionally subjected to selection methods to obtain a homogenouspopulation of iPSC or fully clonal iPSC population, (iii) subjected toselection methods to obtain a specific cell population (e.g. CD34+cells) and then reprogrammed, or (iv) subjected to selection methods toobtain a specific cell population (e.g. CD34+ cells) and subsequentlyexpanded and then reprogrammed. In some aspects of the invention,mononuclear cells are reprogrammed to a pluripotent state. Reprogrammedcells may be cultured under conditions sufficient as disclosed herein toprovide ground state pluripotent cells.

In embodiments of the invention, a specific population of mononuclearcells from the minimal volume of blood is selected for reprogrammingusing methods known in the art, including, but not limited to,fluorescent activated cell sorting (FACS) and magnetic assisted cellsorting (MACS) (e.g. immuno-magnetic bead selection). Non-limitingexamples of mononuclear cells suitable for practicing the inventioninclude, but are not limited to, CD45+/CD34+/Lineage-cells,CD45+/Lineage− cells, hematopoetic stem and progenitor cells (e.g. CD34+cells), myeloid progenitor cells, lymphoid progenitor cells, mast cells,monocytes, lymphocytes, cytotoxic T cells, helper T cells, regulatory Tcells, natural killer T cells, memory T cells, B cells (e.g. plasma Bcells, memory B cells, B-1 cells and B-2 cells), neutrophils,macrophages, basophils, dendritic cells and eosinophils.

In some embodiments of the invention, mononuclear cells are reprogrammedby non-integrative reprogramming. Suitable methods of non-integrativereprogramming for use with the invention include, but are not limitedto, non-integrating lentivirus, Sendai virus, episomal reprogramming,or, introducing mRNA or naked DNA by chemical or electroporationmethodologies or applying reprogramming factors as fusion proteins withprotein transduction domains.

In some embodiments, the invention provides methods for culturing andexpanding mononuclear cells from a minimal volume blood (e.g. umbilicalcord blood, placental blood or peripheral blood). As with otherembodiments of the invention, blood for use with the invention may beobtained from human or non-human sources, such non-human sourcesincluding, but not limited to non-human primate, mouse, rat, horse, pig,bovine and avian. Suitable mononuclear cells for culturing and expandingas disclosed herein include, but are not limited to, non-pluripotentcells, incompletely or partially pluripotent stem cells, multipotentcells, oligopotent cells, unipotent cells, terminally differentiatedcells, or a mixed population of cells comprising any combination of theforegoing. In some aspects, mononuclear cells that are cultured andexpanded include, but are not limited to, hematopoetic stem andprogenitor cells (e.g. CD34+ cells), myeloid progenitor cells, lymphoidprogenitor cells, mast cells, monocytes, lymphocytes, cytotoxic T cells,helper T cells, regulatory T cells, natural killer T cells, memory Tcells, B-cells (e.g. plasma B cells, memory B cells, B-1 cells and B-2cells), neutrophils, macrophages, basophils, dendritic cells andeosinophils. Mononuclear cells expanded and cultured according to theinvention may be used reprogrammed to iPSC, or used for banking (e.g.creating a repository of cells for potential therapeutic use).

A minimal volume of blood for use with the invention may comprise anyvolume that provides a number of mononuclear cells sufficient to achievereprogramming as disclosed herein. Minimal volumes of blood forreprogramming may be between about 50 μl and about 1 ml. Minimal volumesof blood may be about 900 μl, about 800 μl, about 700 μl, about 600 μl,about 500 μl, about 400 μl, about 300 μl, about 200 μl, about 100 μlabout 50 μl, as well as any volume intervening these specificallyspecified volumes. In some embodiments, a minimal volume of blood is anamount of UCB blood provided in a cord blood segment. The minimal volumeof blood may comprise a volume of blood sufficient to contain at leastabout 100 mononuclear cells. The minimal volume of blood may comprise avolume of blood sufficient to contain a number of mononuclear cells thatis about 100, 1,000, 1×10³, 1×10⁴, 1×10⁵ or 1×10⁶ cells, as well as anynumber of cells intervening these amounts. The minimal volume of bloodmay contain less than about 1×10³ mononuclear cells.

In some aspects of the invention, small molecules are used in thereprogramming of mononuclear cells. Accordingly, in particularembodiments, reprogramming mononuclear cells comprises introducing oneor more reprogramming factors into the cells as contemplated herein andcontacting the cells with at least one of a GSK3 inhibitor; a MEKinhibitor; a TGFβR inhibitor, and a Rho Kinase (ROCK) inhibitor. Suchreprogramming procedures may improve the efficiency of reprogrammingmononuclear cells. Improvements in efficiency of reprogramming can bemeasured by (1) a decrease in the time required for reprogramming andgeneration of pluripotent cells (e.g., by shortening the time togenerate pluripotent cells by at least a day compared to a similar orsame process without the small molecule), or alternatively, or incombination, (2) an increase in the number of pluripotent cellsgenerated by a particular process (e.g., increasing the number of cellsreprogrammed in a given time period by at least 10%, 30%, 50%, 100%,200%, 500%, etc. compared to a similar or same process without the smallmolecule). In some embodiments, a 2-fold to 20-fold improvement inreprogramming efficiency is observed. In some embodiments, reprogrammingefficiency is improved by more than 20 fold. In some embodiments, a morethan 100 fold improvement in efficiency is observed over the methodwithout the small molecule reprogramming agent (e.g., a more than 100fold increase in the number of pluripotent cells generated).

In one embodiment, reprogramming mononuclear cells comprises introducingone or more reprogramming factors into the mononuclear cells ascontemplated herein and contacting the cells with a GSK3 inhibitor; aMEK inhibitor; and a TGFβR inhibitor, and optionally a ROCK inhibitor.In another embodiment, reprogramming mononuclear cells comprisesintroducing one or more reprogramming factors into the cells ascontemplated herein and contacting the cells with a GSK3 inhibitor; aMEK inhibitor; a TGFβR inhibitor, and a ROCK inhibitor.

In some aspects, reprogrammed mononuclear cells are cultured in thepresence of one or more of a GSK-3 inhibitor, a MEK inhibitor, andoptionally a ROCK inhibitor, wherein the cell culture medium does notcomprise, or is essentially free of, an inhibitor of TGFβ/activinsignaling pathways, including TGFβ receptor (TGFβR) inhibitors and ALK5inhibitors, as contemplated herein. Without wishing to be bound to anyparticular theory, it is contemplated that long-term culture ofpluripotent cells with a TGFβR/ALK5 inhibitor leads to spontaneousdifferentiation of the reprogrammed mononuclear cells. Thus, culturingreprogrammed mononuclear cells in the presence of a GSK-3 inhibitor, aMEK inhibitor, and a Rho Kinase (ROCK) inhibitor, but not a TGFβR/ALK5inhibitor, may provide ground state pluripotent cells.

In particular embodiments, a mononuclear cell is reprogrammed by themethods disclosed herein and subsequently, the reprogrammed mononuclearcell is cultured to a stable ground state of pluripotency by culturingthe cell in a medium comprising a GSK-3 inhibitor, a MEK inhibitor, anda Rho Kinase (ROCK) inhibitor, wherein the media lacks a TGFβR/ALK5inhibitor.

In some embodiments, a mononuclear cell is reprogrammed by introducingone or more reprogramming factors and culturing the cell in a mediumcomprising a GSK-3 inhibitor, a MEK inhibitor, a Rho Kinase (ROCK)inhibitor, and a TGFβR/ALK5 inhibitor, and subsequently, thereprogrammed mononuclear cell is cultured to a provide cells withreduced spontaneous differentiation by culturing the cell in a mediumcomprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho Kinase (ROCK)inhibitor, wherein the media lacks a TGFβR/ALK5 inhibitor.

One aspect concerns the GSK-3 inhibitors for use with the methods andcompositions disclosed herein. GSK-3β inhibitors are specific exemplaryWnt pathway agonists suitable for use in compositions contemplatedherein, and may include, but are not limited to, polynucleotides,polypeptides, and small molecules. GSK-3β inhibitors contemplated hereinmay decrease GSK-3β expression and/or GSK-3β activity. Illustrativeexamples of GSK-3β inhibitors contemplated herein include, but are notlimited to, anti-GSK-3β antibodies, dominant negative GSK-3β variants,siRNA, shRNA, miRNA and antisense nucleic acids that target GSK-3β.

Other illustrative GSK-3β inhibitors include, but are not limited to:Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, AR-A014418,CT99021, CT20026, SB216763, AR-A014418, lithium, SB 415286, TDZD-8, BIO,BIO-Acetoxime,(5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine,Pyridocarbazole-cyclopenadienylruthenium complex, TDZD-84-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione,2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, OTDZT,alpha-4-Dibromoacetophenone, AR-AO 144-18,3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione;TWSl 19 pyrrolopyrimidine compound, L803 H-KEAPPAPPQSpP-NH2 or itsmyristoylated form; 2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone;GF109203X; RO318220; TDZD-8; TIBPO; and OTDZT.

In particular illustrative embodiments, the GSK-3β inhibitor isCHIR99021, BIO, or Kenpaullone. In a preferred embodiment, the GSK-3βinhibitor is CHIR99021.

An aspect of the invention concerns the MEK inhibitors for use with themethods and compositions disclosed herein. ERK/MEK inhibitors suitablefor use in methods and compositions contemplated herein include, but arenot limited to, polynucleotides, polypeptides, and small molecules.ERK/MEK inhibitors contemplated herein may decrease MEK or ERKexpression and/or MEK or ERK activity. Illustrative examples of MEK/ERKinhibitors contemplated herein include, but are not limited to, anti-MEKor anti-ERK antibodies, dominant negative MEK or ERK variants, siRNA,shRNA, miRNA and antisense nucleic acids that target MEK or ERK.

Other illustrative ERK/MEK inhibitors include, but are not limited to,PD0325901, PD98059, UO126, SL327, ARRY-162, PD184161, PD184352,sunitinib, sorafenib, Vandetanib, pazopanib, Axitinib, GSKl 120212,ARRY-438162, RO5126766, XL518, AZD8330, RDEAl 19, AZD6244, FR180204 andPTK787.

Additional illustrative MEK/ERK inhibitors include those compoundsdisclosed in International Published Patent Applications WO 99/01426, WO02/06213, WO 03/077914, WO 05/051301 and WO2007/044084.

Further illustrative examples of MEK/ERK inhibitors include thefollowing compounds:6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazol-e-5-carboxylicacid (2,3-dihydroxy-propoxy)-amide;6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-pyran-2-ylm-ethyl)-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide,1-[6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimida-zol-5-yl]-2-hydroxy-ethanone,6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazol-e-5-carboxylicacid (2-hydroxy-1,1-dimethyl-ethoxy)-amide,6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-furan-2-ylm-ethyl)-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide,6-(4-Bromo-2-fluoro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazol-e-5-carboxylicacid (2-hydroxy-ethoxy)-amide,6-(2,4-Dichloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylicacid (2-hydroxy-ethoxy)-amide,6-(4-Bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazol-e-5-carboxylicacid (2-hydroxy-ethoxy)-amide, referred to hereinafter as MEK inhibitor1;2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide;referred to hereinafter as MEK inhibitor 2; and4-(4-bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridazine-3-carboxamideor a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the MEK/ERK inhibitor is PD98059.

One aspect of the invention concerns the ROCK inhibitors for use withthe methods and compositions disclosed herein. Rho associated kinases(ROCK) are serine/threonine kinases that serve downstream effectors ofRho kinases (of which three isoforms exist-RhoA, RhoB and RhoC). ROCKinhibitors suitable for use in the methods and compositions contemplatedherein include, but are not limited to, polynucleotides, polypeptides,and small molecules. ROCK inhibitors contemplated herein may decreaseROCK expression and/or ROCK activity. Illustrative examples of ROCKinhibitors contemplated herein include, but are not limited to,anti-ROCK antibodies, dominant negative ROCK variants, siRNA, shRNA,miRNA and antisense nucleic acids that target ROCK.

Illustrative ROCK inhibitors contemplated herein include, but are notlimited to: thiazovivin, Y27632, Fasudil, AR122-86, Y27632 H-1152,Y-30141, Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A, SB-772077-B,N-(4-Pyridyl)-N′-(2,4,6-trichlorophenyl)urea, 3-(4-Pyridyl)-1H-indole,and (R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamideand ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which isherein incorporated by reference in its entirety.

In one embodiment, the ROCK inhibitor is thiazovivin, Y27632, orpyrintegrin.

In a preferred embodiment, the ROCK inhibitor is thiazovivin.

An aspect of the invention concerns the TGFβ receptor (e.g., ALK5)inhibitors for use with the methods and compositions disclosed herein.Suitable TGFβ receptor (e.g., ALK5) inhibitors for use with theinvention can include antibodies to, dominant negative variants of, andantisense nucleic acids that suppress expression of, TGFβ receptors(e.g., ALK5). Exemplary TGFβ receptor/ALK5 inhibitors include, but arenot limited to, SB431542 (see, e.g., Inman, et al., MolecularPharmacology 62(1):65-74 (2002)), A-83-01, also known as3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide(see, e.g., Tojo, et al., Cancer Science 96(11):791-800 (2005), andcommercially available from, e.g., Toicris Bioscience);2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine,Wnt3a/BIO (see, e.g., Dalton, et al., WO2008/094597, herein incorporatedby reference), BMP4 (see, Dalton, supra), GW788388(-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide)(see, e.g., Gellibert, et al., Journal of Medicinal Chemistry49(7):2210-2221 (2006)), SM16 (see, e.g., Suzuki, et al., CancerResearch 67(5):2351-2359 (2007)), IN-1130(3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide)(see, e.g., Kim, et al., Xenobiotica 38(3):325-339 (2008)), GW6604(2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, e.g., deGouville, et al., Drug News Perspective 19(2):85-90 (2006)), SB-505124(2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride) (see, e.g., DaCosta, et al., Molecular Pharmacology65(3):744-752 (2004)) and pyrimidine derivatives (see, e.g., thoselisted in Stiefl, et al., WO2008/006583, herein incorporated byreference). Further, while “an ALK5 inhibitor” is not intended toencompass non-specific kinase inhibitors, an “ALK5 inhibitor” should beunderstood to encompass inhibitors that inhibit ALK4 and/or ALK7 inaddition to ALK5, such as, for example, SB-431542 (see, e.g., Inman, etal., J, Mol. Pharmacol. 62(1): 65-74 (2002). Without intending to limitthe scope of the invention, it is believed that ALK5 inhibitors affectthe mesenchymal to epithelial conversion/transition (MET) process.TGFβ/activin pathway is a driver for epithelial to mesenchymaltransition (EMT). Therefore, inhibiting the TGFβ/activin pathway canfacilitate MET (i.e. reprogramming) process.

In view of the data herein showing the effect of inhibiting ALK5, it isbelieved that inhibition of the TGFβ/activin pathway will have similareffects of inhibiting ALK5. Thus, any inhibitor (e.g., upstream ordownstream) of the TGFβ/activin pathway can be used in combination with,or instead of, ALK5 inhibitors as described herein. ExemplaryTGFβ/activin pathway inhibitors include but are not limited to: TGFβreceptor inhibitors, inhibitors of SMAD 2/3 phosphorylation, inhibitorsof the interaction of SMAD 2/3 and SMAD 4, and activators/agonists ofSMAD 6 and SMAD 7.

TGFβ receptor inhibitors can include antibodies to, dominant negativevariants of and siRNA or antisense nucleic acids that target TGFβreceptors. Specific examples of TGFβ receptor inhibitors include but arenot limited to SU5416;2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride (SB-505124); lerdelimumb (CAT-152); metelimumab (CAT-192);GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580276; LY364947;LY2109761; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894;SB-203580; SD-093; Gleevec; 3,5,7,2′,4′-pentahydroxyflavone (Morin);activin-M108A; P144; soluble TBR2-Fc; and antisense transfected tumorcells that target TGFβ receptors. (See, e.g., Wrzesinski, et al.,Clinical Cancer Research 13(18):5262-5270 (2007); Kaminska, et al., ActaBiochimica Polonica 52(2):329-337 (2005); and Chang, et al., Frontiersin Bioscience 12:4393-4401 (2007).)

Inhibitors of SMAD 2/3 phosphorylation can include antibodies to,dominant negative variants of and antisense nucleic acids that targetSMAD2 or SMAD3. Specific examples of inhibitors include PD169316;SB203580; SB-431542; LY364947; A77-01; and3,5,7,2′,4′-pentahydroxyflavone (Morin). (See, e.g., Wrzesinski, supra;Kaminska, supra; Shimanuki, et al., Oncogene 26:3311-3320 (2007); andKataoka, et al., EP1992360, incorporated herein by reference.)

Inhibitors of the interaction of SMAD 2/3 and smad4 can includeantibodies to, dominant negative variants of and antisense nucleic acidsthat target SMAD2, SMAD3 and/or smad4. Specific examples of inhibitorsof the interaction of SMAD 2/3 and SMAD4 include but are not limited toTrx-SARA, Trx-xFoxH1b and Trx-Lef1. (See, e.g., Cui, et al., Oncogene24:3864-3874 (2005) and Zhao, et al., Molecular Biology of the Cell,17:3819-3831 (2006).)

Activators/agonists of SMAD 6 and SMAD 7 include but are not limited toantibodies to, dominant negative variants of and antisense nucleic acidsthat target SMAD 6 or SMAD 7. Specific examples of inhibitors includebut are not limited to smad7-as PTO-oligonucleotides. (See, e.g.,Miyazono, et al., U.S. Pat. No. 6,534,476, and Steinbrecher, et al.,US2005119203, both incorporated herein by reference.

In some aspects, the invention provides compositions comprising aminimal volume of blood comprising mononuclear cells and at least one ofthe reprogramming factors disclosed herein. The composition may comprisea minimal volume of UCB comprising mononuclear cells and at least onereprogramming factor disclosed herein. The composition may comprise acord blood segment comprising mononuclear cells and at least onereprogramming factor disclosed herein. The composition may comprise aminimal volume of blood comprising mononuclear cells and at least one ofOCT4, SOX2, KLF4 and c-MYC. The composition may comprise a minimalvolume of blood comprising mononuclear cells and at least one of OCT4,SOX2, KLF4 and LIN28. The composition may comprise a minimal volume ofUCB comprising mononuclear cells and at least one of OCT4, SOX2, KLF4and c-MYC. The composition may comprise a minimal volume of bloodcomprising mononuclear cells and at least one of OCT4, SOX2, KLF4 andLIN28. The composition may comprise a minimal volume of blood comprisingmononuclear cells and at least one of OCT4, NANOG, ECAT1, ESRRB, andUTF1. The composition may comprise a minimal volume of UCB comprisingmononuclear cells and at least one of OCT4, NANOG, ECAT1, ESRRB, andUTF1. The composition may comprise a minimal volume of blood comprisingmononuclear cells and at least one of OCT4, SOX2, and SV40LT. Thecomposition may comprise a minimal volume of UCB comprising mononuclearcells and at least one of OCT4, SOX2, and SV40LT. The composition maycomprise a minimal volume of blood comprising mononuclear cells and atleast one of OCT4, ECAT1, and UTF1. The composition may comprise aminimal volume of UCB comprising mononuclear cells and at least one ofOCT4, ECAT1, and UTF1.

In some aspects, the invention provides compositions comprising aminimal volume of blood comprising mononuclear cells, at least one ofthe reprogramming factors disclosed herein, and at least one of a GSK3inhibitor; a MEK inhibitor; a TGFβR inhibitor, and a ROCK inhibitor. Thecomposition may comprise a minimal volume of UCB comprising mononuclearcells, at least one reprogramming factor disclosed herein, and at leastone of a GSK3 inhibitor; a MEK inhibitor; a TGFβR inhibitor, and a ROCKinhibitor. The composition may comprise a cord blood segment comprisingmononuclear cells, at least one reprogramming factor disclosed herein,and at least one of a GSK3 inhibitor; a MEK inhibitor; a TGFβRinhibitor, and a ROCK inhibitor. The composition may comprise a minimalvolume of blood comprising mononuclear cells, at least one of OCT4,SOX2, KLF4 and c-MYC, and at least one of a GSK3 inhibitor; a MEKinhibitor; a TGFβR inhibitor, and a ROCK inhibitor. The composition maycomprise a minimal volume of blood comprising mononuclear cells, atleast one of OCT4, SOX2, KLF4 and LIN28, and at least one of a GSK3inhibitor; a MEK inhibitor; a TGFβR inhibitor, and a Rho Kinase (ROCK)inhibitor. The composition may comprise a minimal volume of UCBcomprising mononuclear cells, at least one of OCT4, SOX2, KLF4 andc-MYC, and at least one of a GSK3 inhibitor; a MEK inhibitor; a TGFβRinhibitor, and a ROCK inhibitor. The composition may comprise a minimalvolume of blood comprising mononuclear cells, at least one of OCT4,SOX2, KLF4 and LIN28, and at least one of a GSK3 inhibitor; a MEKinhibitor; a TGFβR inhibitor, and a ROCK inhibitor. The composition maycomprise a minimal volume of blood comprising mononuclear cells, atleast one of OCT4, NANOG, ECAT1, ESRRB, and UTF1, and at least one of aGSK3 inhibitor; a MEK inhibitor; a TGFβR inhibitor, and a ROCKinhibitor. The composition may comprise a minimal volume of UCBcomprising mononuclear cells, at least one of OCT4, NANOG, ECAT1, ESRRB,and UTF1, and at least one of a GSK3 inhibitor; a MEK inhibitor; a TGFβRinhibitor, and a ROCK inhibitor. The composition may comprise a minimalvolume of blood comprising mononuclear cells, at least one of OCT4,SOX2, and SV40LT, and at least one of a GSK3 inhibitor; a MEK inhibitor;a TGFβR inhibitor, and a ROCK inhibitor. The composition may comprise aminimal volume of UCB comprising mononuclear cells, at least one ofOCT4, SOX2, and SV40LT, and at least one of a GSK3 inhibitor; a MEKinhibitor; a TGFβR inhibitor, and a ROCK inhibitor. The composition maycomprise a minimal volume of blood comprising mononuclear cells, atleast one of OCT4, ECAT1, and UTF1, and at least one of a GSK3inhibitor; a MEK inhibitor; a TGFβR inhibitor, and a ROCK inhibitor. Thecomposition may comprise a minimal volume of UCB comprising mononuclearcells, at least one of OCT4, ECAT1, and UTF1, and at least one of a GSK3inhibitor; a MEK inhibitor; a TGFβR inhibitor, and a ROCK inhibitor.

In some aspects, the invention provides compositions comprising betweenabout 100 to about 1×10⁴ mononuclear cells and at least one of thereprogramming factors disclosed herein. The composition may comprisebetween about 100 to about 1×10⁴ mononuclear cells and at least one ofOCT4, SOX2, KLF4 and c-MYC. The composition may comprise between about100 to about 1×10⁴ mononuclear cells and at least one of OCT4, SOX2,KLF4 and LIN28. The composition may comprise between about 100 to about1×10⁴ mononuclear cells and at least one of OCT4, SOX2, KLF4 and c-MYC.The composition may comprise between about 100 to about 1×10⁴mononuclear cells and at least one of OCT4, SOX2, KLF4 and LIN28. Thecomposition may comprise between about 100 to about 1×10⁴ mononuclearcells and at least one of OCT4, NANOG, ECAT1, ESRRB, and UTF1. Thecomposition may comprise between about 100 to about 1×10⁴ mononuclearcells and at least one of OCT4, NANOG, ECAT1, ESRRB, and UTF1. Thecomposition may comprise about 100 to about 1×10⁴ mononuclear cells andat least one of OCT4, SOX2, and SV40LT. The composition may compriseabout 100 to about 1×10⁴ mononuclear cells and at least one of OCT4,SOX2, and SV40LT. The composition may comprise about 100 to about 1×10⁴mononuclear cells and at least one of OCT4, ECAT1, and UTF1. Thecomposition may comprise about 100 to about 1×10⁴ mononuclear cells andat least one of OCT4, ECAT1, and UTF1.

In some aspects, the invention provides compositions comprising about100 to about 1×10⁴ mononuclear cells, at least one of the reprogrammingfactors disclosed herein, and at least one of a GSK3 inhibitor; a MEKinhibitor; a TGFβR inhibitor, and a ROCK inhibitor. The composition maycomprise about 100 to about 1×10⁴ mononuclear cells, at least one ofOCT4, SOX2, KLF4 and c-MYC, and at least one of a GSK3 inhibitor; a MEKinhibitor; a TGFβR inhibitor, and a ROCK inhibitor. The composition maycomprise about 100 to about 1×10⁴ mononuclear cells, at least one ofOCT4, SOX2, KLF4 and LIN28, and at least one of a GSK3 inhibitor; a MEKinhibitor; a TGFβR inhibitor, and a ROCK inhibitor. The composition maycomprise about 100 to about 1×10⁴ mononuclear cells, at least one ofOCT4, SOX2, KLF4 and c-MYC, and at least one of a GSK3 inhibitor; a MEKinhibitor; a TGFβR inhibitor, and a ROCK inhibitor. The composition maycomprise about 100 to about 1×10⁴ mononuclear cells, at least one ofOCT4, SOX2, KLF4 and LIN28, and at least one of a GSK3 inhibitor; a MEKinhibitor; a TGFβR inhibitor, and a ROCK inhibitor. The composition maycomprise about 100 to about 1×10⁴ mononuclear cells, at least one ofOCT4, NANOG, ECAT1, ESRRB, and UTF1, and at least one of a GSK3inhibitor; a MEK inhibitor; a TGFβR inhibitor, and a ROCK inhibitor. Thecomposition may comprise about 100 to about 1×10⁴ mononuclear cells, atleast one of OCT4, NANOG, ECAT1, ESRRB, and UTF1, and at least one of aGSK3 inhibitor; a MEK inhibitor; a TGFβR inhibitor, and a ROCKinhibitor. The composition may comprise about 100 to about 1×10⁴mononuclear cells, at least one of OCT4, SOX2, and SV40LT, and at leastone of a GSK3 inhibitor; a MEK inhibitor; a TGFβR inhibitor, and a ROCKinhibitor. The composition may comprise about 100 to about 1×10⁴mononuclear cells, at least one of OCT4, SOX2, and SV40LT, and at leastone of a GSK3 inhibitor; a MEK inhibitor; a TGFβR inhibitor, and a ROCKinhibitor. The composition may comprise about 100 to about 1×10⁴mononuclear cells, at least one of OCT4, ECAT1, and UTF1, and at leastone of a GSK3 inhibitor; a MEK inhibitor; a TGFβR inhibitor, and a ROCKinhibitor. The composition may comprise about 100 to about 1×10⁴mononuclear cells, at least one of OCT4, ECAT1, and UTF1, and at leastone of a GSK3 inhibitor; a MEK inhibitor; a TGFβR inhibitor, and a ROCKinhibitor.

All publications, patent applications, and issued patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or issued patent were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The following examples are provided byway of illustration only and not by way of limitation. Those of skill inthe art will readily recognize a variety of noncritical parameters thatcould be changed or modified to yield essentially similar results.

Example 1 CD34+ Cell Selection and Cell Expansion (Including ExpansionMedia)

Blood cells derived from fresh or frozen peripheral or umbilical cordblood collected in minimal volumes, such as 500 or 100 μL from cordblood segments, were processed by either; 1) Ficoll enrichment formononuclear cells followed by hematopoietic stem cell culture andexpansion and subsequent flow cytometry sort for CD45+/CD34+/Lineage−cells prior to reprogramming (FIG. 2A), or 2) initially flow cytometrysorted for CD34+/CD45+/Lineage-population expanded in hematopoietic stemcell culture medium (FIG. 2C) followed by reprogramming. Cells expandedefficiently under the culture conditions (FIG. 2B) and produced ahomogenous population of cells as demonstrated by cell flow profile forLin and CD34 (FIG. 2D). Hematopoietic stem cell media consisted of serumfree base medium SFM (Stem Cell Technologies) supplemented withcytokines stem cell factor (SCF) ranging 10-100 ng/mL, Thrombopoietin(TPO) ranging 10-100 ng/mL, FLT3L ranging 10-100 ng/mL, basic fibroblastgrowth factor (bFGF) ranging 5-50 ng/mL and insuling growth factor (IGF)ranging 5-50 ng/mL. SFM was optionally replaced with Iscoves MEM(Corning) supplemented with serum replacement cocktail orinsulin/transferrin/selenium (Sigma). Collected blood cells wereresuspended in hematopoietic stem cell medium and transferred topreviously matrigel (Corning) coated wells. Every 2 to 3 days theculture received additional fresh medium and pipette up and down tobreak up cell clumps formed during suspended culture proliferation.Cells were readily transfected as demonstrated by GFP expression (FIG.2E).

Example 2 Reprogramming Mononuclear Cells

To initiate reprogramming, ectopic expression of reprogramming factorswas induced by lentiviral transduction or episomal vector transfection.Lentiviral transfection was followed as previously described (Valamehret al. Sci Rep. 2012; 2:213). Briefly, the cells from Example 1 wereplated at 1×10⁵ cells per well of a 6-well plate on Matrigel (BDBiosciences) coated surface. Unless specified, all Matrigel coatingsconsists of adding Matrigel solution (one aliquot of Matrigelresuspended in 25 mL DMEM/F12) to tissue culture surfaces and allowingfor 2-4 hrs incubation at 37° C. Supernatant from 293T cells generatinglentivirus expressing transgene OCT4/SOX2/SV40LT was added to thestarting cells at a dilution of 1:2 (one part lentiviral supernatant:onepart fibroblast medium), supplemented with 4 μg/mL polybrene(Millipore), and transferred to 37° C. and 5% CO₂ for 12-16 hrs. Forepisomal vector reprogramming, transfection of cord blood cells usinggene set OCT4/SOX2/SV40LT (A14703, Life Technologies) was conductedusing NEON Transfection System (Life Technologies). Approximately, 4 μgof vector set was transfected into 2.5×10⁵ cord blood cells usingsettings 1650 v/10 ms/3 pulses in appropriate buffers as described byproduct manual. The transfected cells were plated directly into a 10 cmdish (fibroblast) or a well of 6-well plate (cord blood) coated withMatrigel and containing hematopoietic stem cell medium (see above)supplemented with 10 ng/mL bFGF and 5 μg/mL fibronectin (BDBiosciences). Twenty-four hours post transfection, FRM was added to theculture in equal volume. The culture medium was switched to entirely FRMon day 5 with hygromycin removed on day 7 post transfection. Allreprogramming cultures were switched to FMM on day 14 post transfection.Cell flow cytometry profile demonstrated expression of SSEA4 and TRA181and an iPSC phenotype (FIGS. 3A and 3B). Twenty-four hours posttransfection, FRM was added in equal volume and continuously added everyfew days until day 14 post transfection where the culture was aspiratedand replaced with entirely FMM. Clusters of adherent rounded cells wereseen around days 5 to 7 post transfection. Once in FMM, reprogrammingcultures were maintained and single cell passaged using Accutase oneither Matrigel or Vitronectin coated surface. The single celldissociated cells were expanded onto Matrigel coated plates with FMM andmaintained until flow cytometry sorting. Reprogramming efficiency wasdetermined by cell flow analysis of SSEA4 and TRA181 and colony counts(FIG. 3A), as well the expression of NANOG (FIG. 3B). In Vitronectin(Life Technologies) surface coating studies, all aspects were kept thesame except for the substitution of Matrigel for Vitronectin. Forreduced factor episomal reprogramming, pCEP4 (Life Technologies) vectorbackbone was constructed to contain OCT4-P2A-OCT4, OCT4-P2A-SOX2 orOCT4-P2A-NANOG-T2A-SOX2 under the regulation of EF1α promoter. Thetransfection of reduced factor episomal vectors followed the sameprotocol as described above with the exception of few modifications.EBNA was co-transfected as either EBNA mRNA (20 μg) or vector cassette(2 μg) (Howden et al., 2006). Hygromycin selection was maintained for 10days and FMM was introduced on day 16.

Example 3 H_(I) PSC Maintenance in Small Molecule Culture

Derived hiPSCs according to Example 2 were routinely passaged in feederfree culture as single cells once confluency of the culture reached75-90%. Cells were maintained in FMM. Cultured cells showed expressionof pluripotency markers OCT4, NANOG, TRA181, TRA160, and DAPI (FIG. 4C)and formed a homogenous population of undifferentiated cells asdemonstrated by cell flow detection of NANOG, SSEA4 and TRA181 (FIGS. 4Eand 5A). Over-confluency was seen to exhaust the medium and result indifferentiation. For single cell dissociation, hiPSCs were washed oncewith phosphate buffered saline (PBS) (Mediatech) and treated withAccutase (Millipore) for 3 to 5 min at 37° C. followed with pipetting toensure single cell dissociation. The single cell suspension was thenmixed in equal volume with base medium that does not include smallmolecules serving as a wash medium, centrifuged at 225 g for 4 min,resuspened in FMM and plated on Matrigel coated surface. Passages weretypically 1:6-1:8, transferred tissue culture plates previously coatedwith Matrigel for 2-4 hrs in 37° C. or Vitronectin for 1 hr at 25° C.and fed every two to three days with FMM. Cell cultures were maintainedin a humidified incubator set at 37° C. and 5% CO₂. FMM culture isdiscussed previously (Valamehr et al. Sci Rep. 2012; 2:213)).

TABLE 1 Conventional Fate Fate hESC Reprogramming Maintenance MediumMedium Medium (Conv.) (FRM) (FMM) DMEM/F12 DMEM/F12 DMEM/F12 KnockoutSerum Knockout Serum Knockout Serum Replacement (20%) Replacement (20%)Replacement (20%) N2 (1x) B27 (1x) Glutamine (1x) Glutamine (1x)Glutamine (1x) Non-Essential Amino Non-Essential Amino Non-EssentialAmino Acids Acids Acids β-mercaptoethanol β-mercaptoethanolβ-mercaptoethanol bFGF (10 ng/mL) bFGF (100 ng/mL) bFGF (100 ng/mL) LIF(10 ng/mL) LIF (10 ng/mL) Thiazovivin (5.0 μM) Thiazovivin (5.0 μM)PD0325901 (0.4 μM) PD0325901 (0.4 μM) CHIR99021 (1.0 μM) CHIR99021 (1.0μM) SB431542 (2.0 μM) In combination with Feeder free, in combinationwith Matrigel or MEF Vitronectin

Example 4 Reprogramming of a Minimal Volume of Mononuclear Cells

To initiate reprogramming, ectopic expression of reprogramming factorsis induced by lentiviral transduction using NIL, traditional integratinglentivirus, or electroporation with episomal vectors. As illustrated inFIGS. 7A-B, the lentiviral expression system consists of severalfeatures including an EF1α promoter, specific gene combinations (Table2) and a LOXP site at the 3′ end to allow for CRE-mediated excision ofthe integrated transgenes. Upon CRE-excision, the derived hiPSCs genomeno longer contains transgenes and are essentially footprint-free. Asillustrated in FIGS. 7C-F, the episomal constructs have unique featuresincluding an EF1α promoter and unique reprogramming factors. Upontransfection, the episomal constructs reside in the nucleus and act in atrans-mediated fashion without integrating into the genome.

For lentivirus infection, the starting mononuclear cells orCD45+/CD34+/Lineage− cells are seeded at 100 to 1×10⁴ cells per well ofa 6-well plate coated with Matrigel (Corning) per manufacturer'sinstructions. Fresh lentiviral supernatant from 293T cells is added tothe starting cells at a dilution of 1:2 (one part lentiviralsupernatant: one part fibroblast medium). NIL viral supernatant is usedat a 1× concentration and not diluted. If previously frozen virus isused, it is not diluted and used at a 1× concentration. Viralsupernatants of various factors are combined (Table 2) up to a total of2 mL of media per 6-well. This is supplemented with 5 μg/mL polybrene(Millipore) and 10 mM Hepes (Meditech) followed by spin infection. Sixwell plates are sealed with parafilm and centrifuged at 600 g for 90 minat 32° C. Plates are then transferred to 37° C. and 5% CO₂ incubatorsfor 12-16 hrs. After incubation with lentivirus, the cells are washedwith PBS and the culture medium switched to 50/50 medium containing onepart FRM and one part hematopoietic medium (ISCOVES media +TPO, SCF,FLT3L). The medium is completely switched to FRM between 4 to 6 dayspost infection. FRM consists of conventional medium (described above)supplemented with 5 μM Thiazovivin (synthesized in-house), 0.4 μMPD0325901 (Biovision), 1 μM CHIR99021 (Biovision), 2 μM SB431542(Biovision), 100 ng/mL bFGF (Life Technologies), 10 ng/mL hLIF(Millipore), 1×N2 Supplement (Life Technologies), and 1×B27 Supplement(Life Technologies). Once wells become confluent, cells are passagedonto 10 cm dishes previously coated with Matrigel. Passaging consists ofdissociation with Accutase (Millipore) onto Matrigel coated surface (asdescribed above). Between days 14 and 18 or when iPSC colonies becomepresent, the culture media is switched from FRM to FMM. The single celldissociated cells are expanded onto Matrigel coated plates with FMM andmaintained until flow cytometry sorting.

TABLE 2 Reprogramming Factor Combinations Vector System OCT4-P2A-OCT4ECAT1-P2A-UTF1 OCT4-P2A-OCT4 NANOG-P2A-ESRRB-T2A-LIN28 ECAT1-P2A-UTF1OCT4-P2A-ESRRB OCT4-P2A-NANOG ECAT1-P2A-UTF1 OCT4-P2A-NANOGECAT1-P2A-UTF1

For episomal vector reprogramming, transfection of mononuclear cells orCD45+/CD34+/Lineage− using the plasmids is conducted using the NEONTransfection System (Life Technologies). Approximately, a total of 3 μgof episomal plasmids containing reprogramming factors is co-transfectedwith EBNA (either in the form of mRNA or as a cassette in cloningplasmid pCDNA) into 100 to 1×10⁴ cells using settings 1650 v/10 ms/3pulses in appropriate buffers as described by product manual. Thetransfected cells are seeded directly onto a well of a 6-well platecoated with Matrigel containing cord blood culture medium supplementedwith 4 ng/mL bFGF and 5 μg/mL fibronectin (BD Biosciences) withoutantibiotics. Cord blood culture medium consists of SFMII+CC110 (StemCell Technologies). Twenty-four hours post transfection, FRM is added tothe culture in equal volume. The culture medium is switched entirely toFRM on day 5 with hygromycin removed 7 days post transfection. Allreprogramming cultures are switched to FMM 14 days post transfection.Twenty-four hours post transfection, FRM is added in equal volume andcontinuously added every few days until day 14 post transfection whenthe culture is aspirated and replaced with entirely FMM. Clusters ofadherent rounded cells are seen around 5 to 7 days post transfection.Once in FMM, all reprogramming cultures are maintained and single cellpassaged using Accutase on Matrigel coated surfaces (described above).The single cell dissociated cells are expanded onto Matrigel coatedplates with FMM and maintained until flow cytometry sorting.

Example 5 Real-Time RT-PCR and Fluidigm Analysis

Total RNA of the cells according to Example 2 was isolated using PicoPure RNA Isolation Kit (Life Technologies). Complimentary DNA (cDNA) wasreverse transcribed from 100 ng of isolated total RNA using the iScriptcDNA Synthesis Kit (Bio-Rad). The cDNA was then used forpre-amplification of specific target genes and two reference controlgenes using the TaqMan PreAmp Master Mix Kit (Life Technologies) and a0.2× concentration of pooled TaqMan assays. Specific targetamplification (STA) from cDNA was performed using 14 cycles ofamplification with the standard cycling conditions stated in themanufacturer's protocol. The pre-amplified cDNA reactions (n=48) werediluted 1:5 (in sterile water) and used as template for the real-timequantitative PCR reactions. 48.48 Dynamic arrays (Fluidigm) were loadedusing a NanoFlex IFC Controller MX (Fluidigm) with TaqMan assays loadedin duplicate and real-time reactions were performed using a BioMarkReal-Time PCR System (Fluidigm). Results were analyzed using BioMarkReal-Time PCR Analysis software (Fluidigm). Samples with cyclethresholds (Cts) above 32 were excluded from the calculations. In caseof hESC control analysis, assay replicates were used to determine SEM.Average Cts were calculated using the mean of two reference genes (GAPDHand HPRT1) against the median of six control MEF cell lines (OSK hiPSCson MEF and H1 ESCs). Relative gene expression results are displayed inExcel (Microsoft) in heat map format. Cells cultured in FMM demonstrateda ground pluripotent as shown by a decreased expression of mesodermal,ectodermal and endodermal cell surface markers (see FIGS. 6A-6C).

TABLE 3 FAM-Labeled TaqMan Probes Catalog Number (Life Assay IDTechnologies) Gene Symbol RefSeq Hs00232764_m1 4331182 FOXA2NM_021784.4; NM_153675.2 Hs00173490_m1 4331182 AFP NM_001134.1Hs00171403_m1 4331182 GATA4 NM_002052.3 Hs00751752_s1 4331182 SOX17NM_022454.3 Hs00610080_m1 4331182 T NM_003181.2 Hs00607978_s1 4331182CXCR4 NM_003467.2; NM_001008540.1 Hs00415443_m1 4331182 NODALNM_018055.4 Hs02330075_g1 4331182 MYOD1 NM_002478.4 Hs00240871_m14331182 PAX6 NM_001127612.1 Hs00801390_s1 4331182 TUBB3 NM_001197181.1;NM_006086.3 Hs00374280_m1 4331182 STAT3 NM_139276.2; NM_213662.1;NM_003150.3 Hs04260366_g1 4331182 NANOG NM_024865.2 Hs00602736_s14331182 SOX2 NM_003106.3 Hs00399279_m1 4331182 ZFP42 NM_174900.3Hs01003405_m1 4331182 DNMT3B NM_001207055.1; NM_001207056.1;NM_006892.3; NM_175848.1; NM_175850.2; NM_175849.1 Hs00702808_s1 4331182LIN28A NM_024674.4 Hs99999003_m1 4331182 MYC NM_002467.4 Hs01081364_m14331182 DNMT3L NM_013369.2; NM_175867.1 Hs00360439_g1 4331182 KLF2NM_016270.2 Hs00222238_m1 4331182 OTX2 NM_172337.1; NM_021728.2Hs00242962_m1 4331182 PAX7 NM_001135254.1; NM_002584.2; NM_013945.2Hs00414521_g1 4331182 DPPA2 NM_138815.3 Hs00216968_m1 4331182 DPPA4NM_018189.3 Hs99999905_m1 4331182 GAPDH NM_002046.4 Hs01003267_m14331182 HPRT1 NM_000194.2 Custom-made TaqMan Gene Expression Assays GeneForward Primer Reverse Primer OCT4 GGGTTTTTGG GCCCCCACC GATTAAGTTCCTTTGTGTT TTCA KLF4 AGCCTAAATG TTGAAAACTT ATGGTGCTTG TGGCTTCCTT GT GTT

Example 6 Testing Presence of Transgenes

Genomic DNA of cells according to Example 2 was isolated using QIAamp®DNA Mini Kit and Proteinase K digestion (Qiagen). 100 ng of the genomicDNA was amplified using transgene-specific primer sets (Table 2 below)(Yu et al., 2007) using Taq PCR Master Mix Kit (Qiagen). The PCRreactions were run for 35 cycles as follows: 94° C. for 30 sec(denaturation), 60-64° C. for 30 sec (annealing) and 72° C. for 1 min(extension). Genomic DNA from fibroblasts and hiPSCs generated usinglentiviral methods were used as negative controls. DNA of the episomalconstructs was used as positive control (FIG. 4D).

TABLE 4 Transgene specific primer sets Amplified region Forward ReverseOct4-Oct4 region of CAGGCCCGAAAGAGAAAG GGAGGGCCTTGGAAGCTTAG episomaltransgene CG Oct4-NANOG region of TATACACAGGCCGATGTGTTGACCGGGACCTTGTCTTC episomal transgene GG OCT4-SOX2 region ofGTGGTCCGAGTGTGGTTCTG GTTCTCCTGGGCCATCTTGC episomal transgeneLin28-SV40pA episomal AAGCGCAGATCAAAAGGA CCCCCTGAACCTGAAACATA transgeneGA WPRE lentiviral TGCTTCCCGTATGGCTTTC AAAGGGAGATCCGACTCGTC element TGEBNA1 ATCGTCAAAGCTGCACAC CCCAGGAGTCCCAGTAGTCA AG Human GAPDHGTGGACCTGACCTGCCGTCT GGAGGAGTGGGTGTCGCTGT

Example 7 Immunocytochemistry Analysis

Cells according to Example 2 were fixed using 4% v/v paraformaldehyde(Alfa Aesar), washed three times with PBS containing 0.2% v/v Tween(PBST) (Fisher Scientific) and permeablized using 0.15% v/v TritonX-100(Sigma-Aldrich) in PBS for 1 hr at 25° C. After permeabilization, cellswere blocked with 1% v/v BSA (Sigma) in PBST (PBSTB) (Fisher Scientific)for 30 min at 25° C. After gentle removal of PBSTB, cells were incubatedwith primary antibody in PBSTB overnight at 4° C. Primary antibodiesused in this study include OCT4 (Santa Cruz), NANOG (Santa Cruz), TRA160(Millipore) and TRA181 (Millipore). β-III Tubulin (TUJ1, R&D Systems),α-Smooth Muscle Actin (Sigma), and Alpha-1-Fetoprotein (Dako) (FIG. 5C).After the overnight incubation, cells were washed three times with PBSTand stained with secondary antibody (Alexa Fluor 488 or 555; Invitrogen)diluted 1:250 in PBSTB for 1 hr at 37° C. The cells were washed threetimes in PBST and stained with Hoechst dye (Invitrogen). For H3K27me3staining analysis, hiPSCs were grown 72 to 96 hrs on cover slips andfixed with 4% parafomaldehyde (Electron Microscopy Science, EMS) in PBSfor 15 min at 25° C. (FIG. 6C). Cell permeabilization was performed with0.1% Triton X-100 in PBS for 1 hour at 25° C., and then cells wereincubated with blocking solution (1% BSA in PBS) for 30 min at 25° C.After blocking, cover slips were incubated with 1:1600 dilution ofanti-trimethyl-histone H3 (Lys27) antibody (Millipore 07-449, H3K27me3)in blocking solution, overnight at 4° C. Secondary antibodies were AlexaFluor 555 Goat-anti-Rabbit IgG (Life Technologies, A21429). The nucleiwere counterstained with DAPI and viewed with an Axio Observer InvertedMicroscope (Carl Zeiss). Images were captured with the AxioVS40 v4.8.1.0(Carl Zeiss Imaging Solutions Gmbh).

Example 8 Differentiation Analysis (EB and Directed)

hiPSC according to Example 2 were differentiated as EBs indifferentiation medium containing DMEM/F12 (Mediatech), 20% fetal bovineserum (Invitrogen), 1% non-essential amino acids (Mediatech), 2 mML-glutamine (Mediatech) and 100 μM β-mercaptoethanol. Briefly, for EBformation hiPSCs were seeded in FMM and switched to conventional mediumthe following day to prime the cells. After 3 to 4 days in conventionalmedium, cultures were single cell dissociated with Accutase (Millipore)and resuspended in differentiation medium including 10 μM Y27632 to afinal concentration of 100,000 cells/mL. ROCK inhibitor Y27632 insteadof Thiazovivn was used for EB formation. Cells were seeded at 100μL/well in V-bottom 96-well non-tissue culture plate (Nunc) andcentrifuged at 950 g for 5 min. The following day compact “ball-likeclumps” were transferred to ultra-low binding 6-well plate (Corning)using P1000 at approximately 30-40 EBs/well in differentiation medium.After 7 days, EBs were transferred at 1:1 to Matrigel coated 6-wellplate and fed with differentiation medium every three days. After 3weeks in culture, cells were fixed and stained. For directed monolayerdifferentiation, hiPSCs were seeded on Matrigel coated wells in FMM todeliver 50% and 90% confluency the following day. Both densities wereinduced to differentiate. For neural induction (Lee et al., 2007), FMMmedia was replaced with hESC media supplemented with 10 μM SB431542 and100 nM LDN-193189 (both SMAD inhibitors, Biovision). Following 2 days,differentiation media was supplemented with 3 μM CHIR99021 (Biovision)in addition to the dual SMAD inhibitors. Cells were fixed two days laterand stained for Nestin (Abcam). For mesoderm differentiation, media wasreplaced with RPMI (Mediatech) supplemented with 1×B27 media additive(Life Technologies), 3 μM CHIR99021, 4 ng/ml bFGF and 10 ng/ml BMP4.Media was changed every other day and cells fixed on the 4th day andstained for aSMA (Sigma). Endoderm differentiation was performed usingthe Human Pluripotent Stem Cell Functional Identification Kit (R&DSystems). hiPSCs were incubated with endoderm differentiation media for3 days, fixed and stained for SOX17 (R&D Systems). Cells demonstrated EBformation and stained positive for β-III Tubulin (TUJ1, R&D Systems),a-Smooth Muscle Actin (Sigma), and Alpha-1-Fetoprotein (Dako) (FIG. 5C).

Example 9 Gene Expression Analysis

RNA was extracted using the PicoPure RNA Isolation kit (LifeTechnologies) using the manufacturers recommended protocol. Total RNAwas quantified using the Nanodrop 2000 Spectrophotometer (ThermoScientific). In brief, biotinylated aRNA was prepared from roughly 100ng of total RNA using the standard protocol for MessageAmp II aRNAAmplification Kit (Applied Biosystems/Ambion, Austin, Tex.) utilizingthe optional Second Round Amplification and then transcribed into biotinlabeled aRNA using MessageAmp II Biotin Enhanced Kit (AppliedBiosystems/Ambion, Austin, Tex.) using the standard protocol. Biotinlabeled aRNA was purified and fragmented according to Affymetrixrecommendations. 20 μg of fragmented aRNA were used to hybridize to theHuman Genome U133-plus-2.0 chips (Affymetrix Inc. Santa Clara, Calif.)for 16 hours at 45° C. The arrays were washed and stained in theAffymetrix Fluidics Station 450 and scanned using the AffymetrixGeneChip Scanner 3000 7 G. Raw expression data files are available onGene Expression Omnibus (GSE50868). The image data were analyzed usingAffymetrix Expression Console software using default analysis settings.Arrays were normalized by log scale robust multi-array analysis (RMA,Affymetrix) and visualized in Spotfire for Genomics 4.5 (Tibco Spotfire,Palo Alto, Calif.). Biological pathway enrichment analysis of thedifferentially expressed probes was performed against the Gene Ontology(GO) database (Singular Enrichment to GO Biological Process and p-value<0.01) using Database for Annotation, Visualization and IntegratedDiscovery (DAVID v6.7) (Huang da et al., 2009a, b). Hierarchicalclustering was performed to compare the gene expression profiles betweensamples based on Log 2 expression levels using a complete linkageclustering method with Euclidean distance measurements (Spotfire forGenomics 4.5). Probe sets for clustering were selected by either anoverall differential in expression levels (> or <2.5-fold) or presenceon targeted gene lists defining a ground or metastable state. For XChromosome gene expression comparison, RMA normalized Affymetrix genechip probe set intensities were converted to linear expression values bytaking the 2̂[RMA log 2 intensity]. Linear expression ratios werecalculated as the naïve expression set divided by the primed expressionset. The expression ratios for all 1688 probe sets mapped to the Xchromosome were visualized in Spotfire 4.5 with the probe sets greateror less than 2 fold enrichment ratio highlighted (FIGS. 6A and 6B).

Example 10 Microarray (ACGH+SNP) Analysis

High resolution array comparative genomic hybridization+ singlenucleotide polymorphism (Agilent SurePrint G3 Human Genome CGH+SNPMicroarray Kit, 4×180K; NCBI Build 37/hg19) and subsequent copy numbervariation and loss of heterozygosity analysis was conducted by WiCellCytogenetics (Madison, Wis.). Relative copy number and regions ofhomozygosity was determined by comparative differential hybridization oflabeled genomic DNA to the 180,000 oligonucleotide whole genome tilingarray (FIG. 4B).

Example 11 Karyotype Analysis

Cytogenetic analysis was performed on G-banded metaphase cells by WiCellResearch Institute (Madison, Wis.). Each karyotype analysis includes aminimum count of 20 spreads with analyses expanded to 40 spread countswhen nonclonal aberrations are identified in the first 20 (FIGS. 5A and5B).

Example 12 Teratoma Formation

Single cell dissociated hiPSCs, at concentrations of 0.5 and 3 millioncells per 200 μL solution (100 μL FMM and 100 μL Matrigel) were injectedsubcutaneously into NOD/SCID/γ^(null) mice. After 5-6 weeks (3 millioncells injection) and 7-8 weeks (0.5 million cells injection), teratomaswere harvested in PBS, fixed overnight at room temperature in 4%paraformaldehyde and maintained thereafter in 70% ethanol at roomtemperature for processing. Samples were sectioned and stained withhematoxylin and eosin. Sections were examined, interpreted andphotographed using a Nikon Eclipse TS100 microscope equipped with aNikon DS-Fil camera (FIG. 5D).

Example 13 Conclusions

The above experiments demonstrate that segments of umbilical cord bloodbanks (or peripheral blood) can be reproducibly reprogrammed in fullydefined culture media to derive hiPSC cell culture banks havingpre-characterized HLA types. This strategy provides access to unique HLAtype cord blood units without comprising the banked cord and representsa cost effective strategy to generate allogeneic hiPSC-derivedtherapeutic cells prepared in advance of therapeutic need. Thereprogramming methodologies disclosed herein significantly enhancereprogramming kinetics of non-integrative induction systems, deliveringa consistent and reproducible derivation method without the need foroncogenes such as KLF4 or c-MYC. Expansion of single cell sorted hiPSCsin FMM ensures clonality, pluripotency and genomic integrity of eachhiPSC line. hiPSCs generated from UCB using the methodologies disclosedherein can be banked under cGMP conditions to provide an attractivesource of cells for downstream differentiated cell therapy applications.

1. A method for producing induced pluripotent stem cells (iPSC) fromperipheral or umbilical cord blood comprising: providing a sample ofperipheral or umbilical cord blood comprising mononuclear cells;reprogramming the mononuclear cells to iPSC by introducing to themononuclear cells a composition comprising (i) at least onepolynucleotide selected from the group consisting of OCT4, SOX2 andSV40LT, or (ii) at least one polypeptide selected from the groupconsisting of OCT4, SOX2 and SV40LT.
 2. The method of claim 1, whereinthe composition is free of polynucleotides encoding c-MYC and KLF4 andis free of c-MYC and KLF4 polypeptides.
 3. The method of claim 1,wherein the sample of blood is less than or about 1 mL, 900 μL, 800 μL,700 μL, 600 μL, 500 μL, 400 μL, 300 μL, 200 μL, 100 μL, 50 μL, 25 μL or10 μL; and/or has about 1×10³ to about 1×10⁶ mononuclear cells. 4.-8.(canceled)
 9. The method of claim 1, wherein the mononuclear cells areCD34+ cells or CD45+/Lineage− progenitor cells with or without beingexpanded prior to reprogramming.
 10. (canceled)
 11. The method of claim1, wherein introducing the at least one polynucleotide is accomplishedby non-integrative reprogramming comprising introducing the at least onepolynucleotide by a non-integrating lentivirus, an episome, mRNA, Sendaivirus, miRNA, or self-replicating RNA.
 12. (canceled)
 13. The method ofclaim 1, wherein reprogramming further comprises contacting themononuclear cells with a composition comprising at least one of a GSK3inhibitor, a MEK inhibitor, a ROCK inhibitor, and a TGFβR inhibitor. 14.The method of claim 12, further comprising culturing the reprogrammedmononuclear cells in a composition comprising a Wnt pathway agonist, aMEK inhibitor, and a ROCK inhibitor, but not a TGFβR inhibitor.
 15. Themethod of claim 13, wherein culturing is performed in a cultureenvironment that is free of feeder cells, or is single cellenzymatically disassociated culturing; or is in a defined culturemedium. 16.-17. (canceled)
 18. The method of claim 13, wherein culturingproduces ground state iPSCs.
 19. The method of claim 1, wherein thereprogramming efficiency of the mononuclear cells is at least about0.01%, at least about 0.1%, at least about 1%, at least about 5%, atleast about 6%, at least about 7%, at least about 8%, at least about 9%or at least about 10%.
 20. (canceled)
 21. The method of claim 1, furthercomprising banking the iPSC.
 22. A method of producing inducedpluripotent stem cells (iPSC) from umbilical cord blood comprising:providing a sample of cryopreserved umbilical cord blood comprisingmononuclear cells, wherein the sample is between about 1 mL and about 10μL in volume; contacting the sample with a composition comprising atleast one of a Wnt pathway agonist, a MEK inhibitor, a ROCK inhibitor,and a TGFR inhibitor; reprogramming the mononuclear cells in thecontacted sample to iPSC by introducing to the mononuclear cells acomposition comprising (i) at least one polynucleotide selected from thegroup consisting of OCT4, SOX2 and SV40LT, wherein the polynucleotidecomprises non-integrating lentivirus, an episome or mRNA, or (ii) atleast one polypeptide selected from the group consisting of OCT4, SOX2and SV40LT; wherein the composition is free of polynucleotides encodingc-MYC and KLF4 and is free of c-MYC and KLF4 polypeptides; and culturingthe reprogrammed mononuclear cells as a feeder free, single cell culturein a defined culture medium comprising a Wnt pathway agonist, a MEKinhibitor, and a ROCK inhibitor, but not a TGFβR inhibitor. 23.-34.(canceled)
 35. The method of claim 1, further comprising differentiatingthe iPSC towards a specific cell type and administering thedifferentiated iPSC to a subject in need of cell therapy.
 36. The methodof claim 34, wherein the iPSCs are allogeneic to the subject and are HLAmatched for the subject.
 37. The method of claim 34, wherein the iPSCare differentiated to form myogenic progenitor cells, hematopoietic stemor progenitor cells, neural progenitor cells, cardiomyocyte progenitorcells, hepatocytes, or pancreatic progenitor cells; or wherein the iPSCare differentiated to form T cells, B cells, dendritic cells, NK cells,myoblasts, hepatic cells or neuronal cells.
 38. (canceled)
 39. Themethod of claim 34, wherein the differentiated iPSC cells are modulatedwith small molecules or large molecules prior to administration to thesubject.
 40. The method of claim 38, wherein the differentiated iPSCcells are modulated with a prostaglandin prior to administration to thesubject.
 41. A composition comprising a sample of umbilical cord bloodor peripheral blood having therein mononuclear cells and (i) at leastone exogenous polynucleotide comprising OCT4, SOX2 and SV40LT, or (ii)at least one exogenous polypeptide comprising OCT4, SOX2 and SV40LT. 42.The composition of claim 40, wherein the composition does not compriseexogenous c-MYC and KLF4 polynucleotides or exogenous c-MYC and KLF4polypeptides.
 43. The composition of claim 40, wherein the umbilicalcord blood or peripheral blood is less than or about 1 mL, less than orabout 900 μL, less than or about 800 μL, less than or about 700 μL, lessthan or about 600 μL, less than or about 500 μL, less than or about 400μL, less than or about 300 μL, less than or about 200 μL, less than orabout 100 μL, less than or about 50 μL, less than or about 25 μL or lessthan or about 10 μL in volume; and/or comprises between about 1×10³ andabout 1×10⁶ mononuclear cells.
 44. (canceled)
 45. The composition ofclaim 40, wherein the mononuclear cells are CD34+ cells.
 46. Thecomposition of claim 40, wherein the at least one exogenouspolynucleotide comprises a non-integrating lentivirus, an episome, mRNA,Sendai virus, miRNA, or self-replicating RNA.
 47. The composition ofclaim 40, further comprising at least one of a Wnt pathway agonist, aMEK inhibitor, a ROCK inhibitor, and a TGFβR inhibitor.
 48. Thecomposition of claim 40, further comprising at least one of a Wntpathway agonist, a MEK inhibitor, and a ROCK inhibitor, but not a TGFβRinhibitor.
 49. A composition comprising a sample of about 1×10³ to about1×10⁶ mononuclear cells and (i) at least one exogenous polynucleotidethat encodes an OCT4 polypeptide, a SOX2 polypeptide and/or a SV40LTpolypeptide, or (ii) at least one of an exogenous OCT4 polypeptide, anexogenous SOX2 polypeptide and an exogenous SV40LT polypeptide.
 50. Thecomposition of claim 48, wherein the composition does not comprise anexogenous c-MYC polynucleotide, an exogenous KLF4 polynucleotide, anexogenous c-MYC polypeptide or an exogenous KLF4 polypeptide. 51.-52.(canceled)
 53. The composition of claim 48, further comprising at leastone of a Wnt pathway agonist, a MEK inhibitor, a ROCK inhibitor, and aTGFβR inhibitor.
 54. The composition of claim 48, further comprising atleast one of a Wnt pathway agonist, a MEK inhibitor, and a ROCKinhibitor, but not a TGFβR inhibitor.
 55. A method of culturingmononuclear cells comprising: providing a minimal volume ofcryopreserved umbilical cord blood or peripheral blood; selectingmononuclear cells from the cord blood or peripheral blood; culturing themononuclear cells under conditions suitable to expand the mononuclearcells.
 56. The method of claim 54, wherein selecting is accomplished byFicoll gradient, fluorescent assisted cell sorting (FACS) or magneticassisted cell sorting (MACS).
 57. The method of claim 54, wherein themononuclear cells are hematopoietic stem or progenitor cells. 58.(canceled)
 59. The method of claim 54, wherein culturing is performedunder defined culture conditions.
 60. The method of claim 56, furthercomprising administering the hematopoietic stem and progenitor cells toa subject in need of hematopoietic reconstitution.
 61. The method ofclaim 59, wherein the hematopoietic stem and progenitor cells areallogeneic to the subject and HLA matched to the subject.
 62. The methodof claim 54, wherein the minimal volume of the umbilical cord blood orperipheral blood is less than or about 1 mL, 900 μL, 800 μL, 700 μL, 600μL, 500 μL, 400 μL, 300 μL, 200 μL, 100 μL, 50 μL, 25 μL or 10 μL;and/or wherein the umbilical cord blood or peripheral blood has betweenabout 1×10³ and about 1×10⁶ mononuclear cells. 63.-64. (canceled) 65.The method of claim 54, further comprising reprogramming the mononuclearcells to pluripotent cells by contacting the mononuclear cells with acomposition comprising (i) at least one polynucleotide that encodes atleast one polypeptide selected from the group consisting of an OCT4polypeptide, a SOX2 polypeptide and a SV40LT polypeptide, or (ii) atleast one polypeptide selected from the group consisting of a OCT4polypeptide, a SOX2 polypeptide and a SV40LT polypeptide.
 66. The methodof claim 64, wherein reprogramming comprises contacting the mononuclearcells with a composition comprising at least one of a GSK3 inhibitor, aMEK inhibitor, a ROCK inhibitor, and a TGFβR inhibitor.
 67. The methodof claim 64, further comprising culturing the pluripotent cells as afeeder free culture, enzymatically passed single cell culture in adefined culture medium that comprises a Wnt pathway agonist, a MEKinhibitor, and a ROCK inhibitor, but not a TGFβR inhibitor.
 68. Themethod of claim 54, wherein the composition does not comprise apolynucleotide encoding a c-MYC polypeptide, a polynucleotide encoding aKLF4 polypeptide, a c-MYC polypeptide or a KLF4 polypeptide.